Rib head dislocation (RHD) in dystrophic scoliosis of type 1 neurofibromatosis (DS-NF1) is a unique disorder caused by skeletal dystrophy and scoliotic instability. No particular surgical manipulation is mentioned in the literature to instruct the spine surgeons to effectively obtain more migration of the dislocated rib head without resection. The present study aimed to investigate the effectiveness of screw/hook insertion at vertebrae with RHDs on the retraction of penetrated rib head from spinal canal.37 neurologically intact patients with DS-NF1 and concomitant 53 RHDs undergoing scoliosis surgery without rib head excision were retrospectively reviewed. We used pre and postoperative whole-spine radiographs to determine the Cobb angle and the vertebral translation (VT), and the CT scans to evaluate the intraspinal rib length (IRL) and rib-vertebral angle (RVA). The dislocated ribs were assigned into two groups according to the presence of screw/hook insertion at vertebrae with RHD: screw/hook group and non-screw/hook group.p = 0.003; 61.8 ± 18.8% vs. 35.1 ± 16.6%, p = 0.001; respectively). Similarly, more correction rates of IRL and RVA were found in the screw/hook group than the non-screw/hook group . Multiple linear regression analysis revealed that the correction rates of Cobb angle, VT and RVA contributed significantly to correction of IRL .37 dislocated ribs with screws/hooks insertion at corresponding vertebrae were assigned into the screw/hook group and the remaining 16 dislocated ribs consisted of the non-screw/hook group. In the screw/hook group, the correction rates of Cobb angle and VT were significantly higher than the non-screw/hook group after surgery (58.7 ± 16.0% vs. 30.9 ± 12.4%, Screw/hook insertion at dystrophic vertebrae with RHDs contributed significantly to the degree of retraction of penetrated rib head from spinal canal. This effectiveness is mediated by more corrections of VT and RVA. Rib head dislocation (RHD) and invasion into the spinal canal is not a rare characteristic of dystrophic scoliosis secondary to type 1 neurofibromatosis (DS-NF1), with an incidence ranging from 12.1–15.9% in the literatures . The potHaving been well documented in previous reports, RHD uniformly occur on the convex side of the scoliosis and was combined with rib penciling , enlargeCurrently, no quantitative analysis was performed regarding whether screw/hook placement in the vertebrae with RHD could be an optimal strategy for retracting the dislocated rib head from the spinal canal. This study is designed to amend this issue, so as to provide detailed data needed to determine whether or not to the fixation placement in dystrophic pedicle is beneficial and should be tried with utmost effort in case of RHD with impending neurological deficits.This is a retrospective study approved by the Institution Review Board of our hospital. Patients with dystrophic scoliosis secondary to NF-1 who underwent corrective scoliosis surgery between March 1998 to January 2018 were identified from our scoliosis database. The following inclusion criteria were used: (1) thoracic scoliosis with typical dystrophic radiographic features [1. Intraspinal rib length . The CT scans were used to analyze and assess the variations of the positional rib-canal interrelationships caused by corrective maneuvers. The parameters for positional assessments were measured pre- and post-operatively as follows: (1) intraspinal rib length ; (2) theParameters were measured on axial CT scans (parameters 1–4) or the standing posteroanterior radiographs (parameters 5 and 6) using the PACS  workstation . Pre- and postoperative CT scans in supine position were obtained in all patients, which were performed preoperatively to provide anatomical information on the severity of the deformed bony vertebral structure to guide the selection and insertion of appropriate pedicle screws, and postoperatively to detect any mispositioned pedicle screws potentially causing neural or vascular injury or impingement. All the patients and their parents were informed about the purpose of CT scans and the potential risks, who then signed informed consent forms. The selection of measurement level on CT scan was identified as the rib head tip with maximal rib penetration and intrusion at the level of intervertebral foramina, both pre- and post-operatively. Two of the well-trained authors (S.L. and Y.Y.M.) completed the measurement individually. In addition, all parameters were selected to determine the inter- and intra- observer variability of the measurement. All the radiographic parameters were measured by the authors and then repeated twice. There were strong inter-observer and intra-observer agreements for all the parameters with all the kappa correlation coefficients exceeding 0.8. Therefore, the measured data were highly reliable, and the mean values of the parameters measured by the two investigators were recorded.t test. Normal distribution test revealed that all p values were > 0.05 in preoperative and postoperative data, or data from groups with or without screw/hook insertion, which indicated that all the data included for the t test were within the normal distribution. Paired-sample t test was applied to compare the operative changes in each group. In addition, the data were compared between the groups with the use of independent-sample t. Multiple linear regression analysis was used to explore whether correction of Cobb angle, vertebral translation, and rib-vertebrae angle could contribute to the extraction of intra-canal rib heads. The significance level was < 0.05.Data analysis was conducted using SPSS 19.0 software (IBM). Shapiro–Wilk test was firstly used to check for the normal distribution of data before performing the Totally 37 patients  with a mean age of 14.8 ± 5.3 years  at surgery were recruited. The intraspinal RHD uniformly located on the convexity of the curve apex regions. Single-level RHD accounted for the largest share , followed by double RHD  and triple RHD . Accordingly, 53 dislocated rib heads were finally recruited and analyzed, and their location was categorized as apex-1 level for 10 cases (19%), apex level for 23 cases (43%), apex + 1 level for 19 cases (36%), and apex + 2 level for 1 case (2%) (Table p < 0.001)  . The height of main curve was improved from 90.0 ± 23.6 mm preoperatively to 103.2 ± 27.0 mm postoperatively, though no statistically significant difference was detected (p = 0.116)  at corresponding RHD levels were inserted into the corresponding vertebral bodies. No neurological deficit was complained after surgery.p = 0.008; 17.6 ± 9.7% vs. 7.2 ± 3.6%, p = 0.006; 24.4 ± 11.0% vs. 11.9 ± 11.3%, p = 0.007). Similarly, on the standing posteroanterior radiographs, the correction rates of main curve and vertebral translation were significantly higher in screw/hook group than non-screw/hook group    Table . The equMultiple challenges of deformity correction have been well documented in treating the complicated DS-NF1, including the management of dislocated rib head penetrating into spinal canal. The neurologic status and the severity of intra-spinal occupation by rib head are the pivotal factors that determine the treatment strategy for this condition . No repoPreviously, resecting the dislocated rib head should be aggressively performed when the following two aspects were indicated , 7, 8. FThe above-mentioned management strategies were apposite for patients with existing or impending neurological impairment, while for patients who were neurologically intact, whether or not to resect the dislocated rib head was still in controversial. The current mainstream perspective was more inclined to retain the dislocated rib head to avoid interfering the spinal cord, tearing the dural sac, and reducing the bleeding and operative time , 10. MaoTechnically, screw insertion at the pedicle was relatively challenging compared with hook placement at the lamina. The dystrophic and thin pedicles brought about significant challenge of reliable pedicle screw insertion, high frequency of screw malposition  and highThe result of the present study showed a rate of 50.9% (27/53) for pedicle screw placement in the corresponding dystrophic vertebrae with dislocated rib head. And the correction rates of Cobb angle, IRL, VT and the RVA in the screw/hook group were significantly higher than those in the non-screw/hook group. Further, more corrections of Cobb angle, VT and the RVA contributed significantly to the correction of IRL through multiple linear regression analysis. The above results confirmed that after screw/hook placement combining with higher corrections of Cobb angle, VT and the RVA at vertebrae with RHD, the three-dimensional spatial relationship between rib head and spinal canal could be changed via translational traction and derotational withdrawal. This was helpful for maximizing the degree of spontaneous withdrawal of dislocated intracanal rib head. Accordingly, if possible, the screw/hook placement at vertebrae with RHD should be aggressively advocated. With the extensive clinical application of O-arm navigation, the precise screw placement will be further increased . This waThe clinical relevance of the current study is that despite being risky and challenging, screw/hook placement at dystrophic vertebrae with RHD was beneficial for spontaneous retracions of the intracanal rib head. For patients without neurological symptoms, preoperative pedicle, lamina and vertebral body morphology at the corresponding level of RHD should be carefully evaluated, which is crucial for assessing the possibility and risk in apical fixation. Navigation-based technique can be used to improve the accuracy of pedicle screw placement. If the pedicle at corresponding level of RHD is not a good recipient to well contain the pedicle screw, the ‘in–out-in’ technique is applicable . OtherwiThe limitations of this study should be addressed. First, CT evaluations at the long-term follow-up were not available currently. Thus, whether or not the correction of the extracted rib head would be lost due to the loss of main curve correction, formation of pseudarthrosis or failure of internal fixation could not be precisely elucidated. Second, it was impossible to tell the difference between screw and hook insertion on degree of withdrawal of rib head due to the limited sample size.Screw/hook insertion at the apical vertebra with RHD is beneficial for retracting the corresponding rib head from spinal canal in DS-NF1. This effectiveness is dependent more on higher corrections of VT and RVA, and could be enhanced by posterior release using posterior column osteotomy.


Response inhibition, whether reactive or proactive, is mostly investigated in a narrow cognitive framework. We argue that it be viewed within a broader frame than the action being inhibited, i.e., in the context of emotion and motivation of the individual at large. This is particularly important in the clinical domain, where the motivational strength of an action can be driven by threat avoidance or reward seeking. The cognitive response inhibition literature has focused on stopping reactively with responses in anticipation of clearly delineated external signals, or proactively in limited contexts, largely independent of clinical phenomena. Moreover, the focus has often been on stopping efficiency and its correlates rather than on inhibition failures. Currently, the cognitive and clinical perspectives are incommensurable. A broader context may explain the apparent paradox where individuals with disorders characterised by maladaptive action control have difficulty inhibiting their actions only in specific circumstances. Using Obsessive Compulsive Disorder as a case study, clinical theorising has focused largely on compulsions as failures of inhibition in relation to specific internal or external triggers. We propose that the concept of action tendencies may constitute a useful common denominator bridging research into motor, emotional, motivational, and contextual aspects of action control failure. The success of action control may depend on the interaction between the strength of action tendencies, the ability to withhold urges, and contextual factors. Effective stopping of maladaptive or inappropriate actions is closely linked to behavioural control and has long been of great interest to clinicians, cognitive psychologists, and neuroscientists. Inhibition of responses is assumed to contribute to adaptive, goal-directed behaviour. As such, it appears to be a useful construct with considerable face validity, often consisting of a clearly observable and intentional suppression of a planned, already initiated, or on-going action. Stopping of actions also appears in the conceptualisation of control processes such as executive functioning , and in The importance of volitional behavioural and cognitive inhibitory control to clinical practice is apparent from inspection of the clinical criteria of mental health conditions. The DSM-5, one of the gold-standard diagnostic systems, outlines a list of criteria for each diagnosis, where patients typically must fulfil a minimum set out of a list of possible criteria over a given time period . There aTaken together, from a clinical perspective, it is clear that whilst difficulties inhibiting responses are relevant to various disorders, the manifestations and instantiations in everyday life are varied and multifaceted. Moreover, whilst almost all individuals can exercise control and effective inhibition in some circumstances, it is the chronic, distressing, and impairing nature in everyday life attributable in part to behavioural disinhibition that characterise those who meet diagnostic criteria. Theorising of several disorders has consequently posited a central role for failures of cognitive control, and more specifically behavioural inhibition. In the case of OCD, inhibition difficulties have been proposed to underly many of the symptoms and neurocognitive findings . SimilarThe presence and centrality of poor inhibitory control across distinct mental health conditions has contributed to its conceptualisation as a transdiagnostic construct . That isIt is important to bear in mind that from a clinical perspective, difficulties in response inhibition are never considered to be the only cognitive impairment in a given disorder but operate as part of a range of general control-related issues . InhibiCentral to advances in the conceptualisation of response inhibition as described thus far has been the adoption of paradigms such as the stop signal task (SST) and go/no-go (GNG) task. Outside of clinical practice which relies on interview, observational, and self-report practices, these have allowed for individual-specific empirical measures of inhibiting prepotent responses in controlled settings. GNG tasks present go and no-go stimuli, requiring participants to respond to the former and withhold responding to the presentation of the latter, at times being viewed as response suppression. In contrast, stop signal tasks (SST) require participants to make a quick response to go stimuli presented on all trials but to countermand or inhibit these prepotent responses when stop signals are occasionally presented immediately after a go stimulus, thus being viewed as response cancellation . The delThese inhibition tasks allow the measurement of response execution in the form of latencies to the go stimuli and their accuracy, but crucially also provide assessment of stopping performance, and as such are versatile in offering measures of multiple related outcome measures. In GNG, this is often in the form of commission errors. SST can also provide the proportion of stop trials where a participant failed to inhibit, but this is qualified by the specific delay values and the staircase procedure, which aims at achieving a success rate of approximately 50% to allow for estimation of stopping latencies. In any case, using the full distribution of go latencies alongside the rates of failed and successful stopping allows for the estimation of the time required to inhibit a response . AlthougThough the use of response inhibition tasks, and SST in particular, is flourishing in clinical research , there aThere is considerable value in investigating stopping behaviourally from a clinical perspective. The stop-signal in particular holds appeal in this sense as it appears straightforward to administer to healthy individuals and patients alike in a relatively brief period of time. Moreover, task instantiation and administration may appear relatively uniform across studies, presumably yielding a comparable general marker of inhibition . At the same time, the task can also be easily adapted to different purposes. However, it is important to consider the goal of any particular study. For example, studies commonly use cross-sectional design to establish whether a particular group, such as those with OCD, exhibit worse stopping performance as measured by SSRT compared to a control group, attributing this to differences in the effectiveness of suppressing responses. To date, such studies have focused largely on reactive stopping, noting medium effects, though against a background of considerable heterogeneity within groups and between studies ,16. ThesAs with all clinical research, any stopping impairments relating to a particular condition or their absence and the heterogeneity within patient groups must be interpreted in light of medication status and comorbidities ,36,37. CA further challenge is that interpretation of findings from a clinical perspective is still far from straightforward. Even if there are robust response inhibition deficits in the majority or a proportion of patients, what may this mean? The presence of difficulties in stopping in cross-sectional correlational designs does not indicate a causal role in the development or maintenance of a disorder such as OCD . For exaThere is another underlying factor playing a role in the seeming discrepancies between the empirical response inhibition research and its clinical conceptualisation. On the one hand, clinical observations of OCD involve dramatic failures of control, with patients unable to refrain from performing rituals for up to several hours daily. This is not commensurable with the medium effect sizes reported above. A key issue that speaks to this is the relatively impoverished contexts in which countermanding and stopping are typically assessed . This haOne approach to address inhibitory control under such particular contexts is to introduce stimuli that are expected to elicit arousal or negative affect, as task-relevant or irrelevant. Numerous studies in non-patient samples have pointed to the deleterious effects of task-irrelevant negative emotion and arousal, presumably resulting from increased attention and processing of the stimuli ,49,50,51A different approach explores how motivation may affect response inhibition by introducing incentives  to standard stopping paradigms. In non-patients samples, it is evident that this can lead to complex strategic interactions, with participants adjusting responding to optimise performance given the modified task constraints . StoppinTheories of emotion typically recognise the interlinked nature of emotion, motivation, and action ,63. ActiTaken together, the introduction of affective or motivationally relevant stimuli or contexts in response inhibition tasks takes steps towards greater clinical relevance, though often the interpretation of such findings can be more challenging. They also provide one strategy to bridging the gap between basic cognitive laboratory findings and everyday behaviour . CarefulTaken together, basic cognitive approaches can offer insight into clinically relevant conditions but appear insufficient in advancing our understanding of the role response inhibition is playing in conditions such as OCD. Intriguingly, theorising and evidence from cognitive-behavioural appraisal accounts of OCD have focused on proactive rather than reactive control. According to such accounts, compulsive behaviours are triggered by normally occurring intrusive thoughts that are catastrophically interpreted as signifying harm and responsibility for its prevention . CompulsSeveral beliefs have been suggested as etiological to the onset and maintenance of symptoms, or the motivation for proactive control. These include: (1) inflated responsibility, (2) over-importance of thoughts, (3) excessive concern about the importance of controlling one’s thoughts, (4) overestimation of threat, (5) intolerance of uncertainty, and (6) perfectionism . HoweverIn order to advance research, it may be useful to examine the seemingly key paradox that individuals with OCD present with: they desire to control and inhibit unwanted thoughts or urges but finding themselves being controlled by their recurrent compulsive behaviours and rituals. It may be the case that OCD patients demonstrate difficulties in the regulation of proactive control. Thus, they exhibit a general tendency of increased effort for proactive control, which is not relaxed even under circumstances where context suggests it beneficial to do so. There are various findings supporting this view. For example, in the Stroop task, OCD patients do not relax control even when neutral trial percentage is increased . AnotherRelatively little research has been conducted on assessing the strength of response in OCD, which may be more closely related to the phenomenology of the disorder in the context of failed inhibition. The construct of action tendencies may be useful in understanding increased urges to respond in OCD, in what is ultimately a maladaptive manner. According to Gibson’s affordances theory , when weIf research supports this line of theorising, it may not only shed light on the underlying cognitive and psychological processes involved in OCD but also facilitate the development of more effective interventions. New interventions are needed because despite the success of existing first-line treatments, including exposure and response prevention and serotonergic medications, approximately half of patients do not show adequate response, with additional patients at a substantial risk of relapse . ChanginIn sum, we argue that to date, response inhibition paradigms such as the SST and GNG have been used to focus on reactive control, while proactive control failures may instead by more central in many disorders. Moreover, although research has often focused on group differences under impoverished contexts, clinical consideration suggests the need to further explore the specific circumstances under which patients experience failures of control, incorporating affective, motivational, and contextual factors. This alongside greater theoretical clarity as to what the role of any observed impairments may mean, will allow a more effective bridging between such lines of research and our understanding of symptoms as experienced in the everyday by patients. Though we present the arguments above with a focus on OCD, they are directly applicable to other mental health conditions, particularly those characterised by excessive impulsivity or compulsivity such as stimulant drug use. Further, there is also benefit in contrasting and comparing findings between disorders. We advocate for a close understanding of the issues faced by patients alongside careful appreciation of the cognitive theory and the strengths and limitations of different methodological approaches. Ultimately, given the complexity and richness of patient experiences, viable and useful advances will require a community of researchers and clinicians working together.


Post-translational modifications such as ubiquitination play important regulatory roles in several biological processes in eukaryotes. This process could be reversed by deubiquitinating enzymes (DUBs), which remove conjugated ubiquitin molecules from target substrates. Owing to their role as essential enzymes in regulating all ubiquitin-related processes, the abundance, localization, and catalytic activity of DUBs are tightly regulated. Dysregulation of DUBs can cause dramatic physiological consequences and a variety of disorders such as cancer, and neurodegenerative and inflammatory diseases. Multiple factors, such as transcription and translation of associated genes, and the presence of accessory domains, binding proteins, and inhibitors have been implicated in several aspects of DUB regulation. Beyond this level of regulation, emerging studies show that the function of DUBs can be regulated by a variety of post-translational modifications, which significantly affect the abundance, localization, and catalytic activity of DUBs. The most extensively studied post-translational modification of DUBs is phosphorylation. Besides phosphorylation, ubiquitination, SUMOylation, acetylation, oxidation, and hydroxylation are also reported in DUBs. In this review, we summarize the current knowledge on the regulatory effects of post-translational modifications of DUBs. Ubiquitination is an essential post-translational protein modification mediated by the ubiquitin (Ub)-conjugating system, which is composed of a Ub-activating enzyme, E1, Ub-conjugating enzyme, E2, and Ub ligase, E3 . The humUbiquitination is a reversible process because the conjugated Ub molecule can be trimmed away from the target protein by deubiquitination enzymes (DUBs). The human genome encodes several kinds of DUBs, which can be divided into seven subfamilies . Among tThe human genome encodes a relatively small number of DUBs compared to Ub ligases; multiplicity does not seem to exist in DUBs. However, a large amount of regulatory mechanisms precisely expand the functions of DUBs in various Ub-related processes to ensure accurate biological responses . The regWith the advancement of analytical tools such as mass spectrometry, PTM sites of DUBs have been identified. Recently, the regulation of DUBs by PTMs and its physiological relevance have been revealed. Studies showed that PTMs can regulate the function of DUBs by altering factors such as its stability, localization, abundance, and catalytic activity, in addition to its involvement in the cell signalling pathway . Here, wThe most straightforward mechanism affecting the biological function of a given protein is its intracellular concentration. Like other signalling proteins, this fact is also applicable to DUBs. The quantity of DUBs is cell or tissue-specific; UCHs subfamily of DUBs are highly abundant in neurons . In contPhosphorylation is an important kind of PTM that influences the essential physiological role of DUBs, and exerts its effect by elevating, stabilizing, or reducing its abundance. Phosphorylation can elevate DUB protein levels by altering their self-assembly and interaction with other partners. Results have shown that AKT-mediated phosphorylation of USP4 at the Ser445 residue is essential for it to form a complex with itself or with other protein partners, such as USP15 or TβRI . In partFurthermore, protein kinase CK2-induced phosphorylation of USP7 at the Ser18 residue plays a major role in maintaining the stability of USP7 protein . PhosphoOn the contrary, phosphorylation can also reduce DUB protein levels by altering the degradation pathway of DUBs. For instance, SYK-dependent phosphorylation of USP25 at residue Tyr740 can sharply reduce its protein levels. This is not caused by proteasome-dependent degradation because addition of a proteasome inhibitor did not rescue USP25 proteins levels . TherefoUbiquitination and deubiquitination are two types of important PTMs. Usually, DUBs play a critical role in the Ub proteasome system (UPS) by deubiquitinating the protein substrate . InteresThe subcellular localization of DUBs is also a key factor in determining the function of DUBs . If bothIndeed, the subcellular localization of some DUBs is tightly controlled so as to facilitate its biological functions. In previous studies involving systematic large-scale proteomic analyses, the subcellular distribution of GFP-labelled DUBs was analysed in HeLa cell lines  Figure . DUBs suMore interestingly, recent studies have shown that the localization of DUBs in cells can be dynamically regulated to facilitate their complex biological functions . For exaPhosphorylation can also affect the function of various DUBs by altering its localization or interactome. Phosphorylation can cause some DUBs to be exported from the nucleus . For insvia Mdm2. Therefore, phosphorylated USP10 can inhibit the growth of tumour cells without inducing mutations in p53, which implies that phosphorylation of USP10 has a potential therapeutic effect against tumours . The complex comprising of USP1 and UAF1 was localized in the cytoplasm. After phosphorylation of USP1, the complex translocated to the nucleus, where the recruitment of the Fanconi anemia protein FANCD2 and PCNA substrates mediated by a SUMO-like domain occurs in response to DNA damage .Similarly, DNA damage-induced ABL1/c-Abl (ABL proto-oncogene 1) activation can promote the phosphorylation of OTULIN at Tyr56, which enhances its interaction with β-catenin and blocks its binding to LUBAC . Then, aFurthermore, the kinase Aurora B plays an important role in mitosis. The protein level of Aurora B varies during the course of cell division. The abnormal regulation of Aurora B during interphase leads to cell cycle defects, which are usually associated with aberrant chromosomal condensation and segregation . StudiesThe receptor-associated protein 80 (RAP80) can recruit BRCA1 to DNA double-strand breaks and induce a DDR . Recent Moreover, a recent study also found that CDC-like kinase 3 (CLK3) or the cholangiocarcinoma-associated CLK3-Q607R mutant can directly phosphorylate USP13 at Tyr708, and promote its binding to c-Myc  Figure . TherefoUbiquitination plays an essential role in numerous cellular processes, such as protein degradation, cell cycle, and transcriptional regulation . SimilarJosephin domain containing 1 (JosD1), a DUB of the MJD subfamily, mainly associates with the cytoskeleton. However, when ubiquitination occurs on JosD1, it tends to localize on the cell membrane  and playFurthermore, BAP1, a member of the UCHs subfamily, is predominantly located in the nucleus and functions as a tumour suppressor . The lysSimilar to phosphorylation of DUBs, ubiquitination can also change the localization of DUBs, which can be detected by changing the interaction partners of DUBs. However, auto-deubiquitination, which is dependent on the catalytic activity of DUBs, can counteract the effect of ubiquitination modification and promote the interaction between DUB and its substrates. For instance, USP4 can be ubiquitinated at multiple sites such as the cysteine residues. Auto-deubiquitination can also occur at these sites, which is required for USP4 to interact with CtIP/MRN and promote DNA repair . MoreoveHypoxia is a commonly encountered physiological stress that can induce an active response in mammalian cells through a transcription factor named hypoxia-inducible factor (HIF) . Oxygen-DUBs are active and substrate-specific enzymes. However, certain DUBs require Ub-binding or modulation to form their active conformation. In a cellular environment, DUB activity is tightly regulated because uncontrolled activation can be detrimental for cells . Herein,Phosphorylation is a central PTM that can regulate the function of a variety of DUBs by directly influencing its catalytic activity . PhosphoE. coli is inactive, but can be activated when the Ser177 residue of OTUD5 is phosphorylated by CKII instead of by mimic phosphorylation  and by-products of mitochondrial oxidative metabolism are continuously produced in eukaryotic cells . Deregul2O2, the deubiquitinase activity was completely abolished as oxidation occurs on cysteine residues in the catalytic domain  samples and cell lines, increased IKK-induced CYLD phosphorylation was observed. Both IKK inhibitors and overexpression of kinase-inactive TBK/IKK can lower CYLD phosphorylation and trigger cell death . These rvia affecting the recruitment of tri-snRNP, suggesting that SUMOylation of USP39 has an essential role in cancer therapy  response by negatively regulate NF-κB activity and stabilize the forkhead box p3 (FOXP3) respectively (H1) and TH17 response  signaling pathway . Similarm Parkin . USP14 cm Parkin . Moreovem Parkin . Study am Parkin . Howevervia allosteric regulatory effects that lead to conformation changes or by exposing a new binding site by abolishing original protein-protein interactions (Generally, single PTMs regulate the function of DUBs ractions . For exaMoreover, complex crosstalk between post-translationally modified proteins often occurs; a well-described example are kinases. Under certain conditions, phosphorylation is often necessary to trigger subsequent phosphorylation, ubiquitination, or SUMOylation, and the ubiquitination or methylation of histones may be essential for its acetylation . The croRecent literature clearly highlights the importance of PTMs in modifying the function of DUBs, and its role in promoting or decreasing the occurrence of diseases. PTMs are therefore emerging as a pivotal regulator of DUBs and may provide novel insights toward the biological functions of DUBs. However, the biological role of PTMs on DUBs has not been fully understood yet, and this requires further research. Structural studies will be particularly important in elucidating the biological role of PTMs in DUBs. There is no doubt that these studies will drive DUB-targeted drug discovery .Recently, several small molecule inhibitors targeted toward different members of DUB subfamilies have been reported. Until now, there are few target-specific inhibitors that have been found . In the Moreover, several DUBs have been reported to be overexpressed or mutated in cancer resulting in altered activities. As PTMs can regulate the abundance and activity of DUBs, it may serve as an effective target for novel cancer therapeutic approaches. We anticipate that the outcome of DUB-focused regulatory research will help decipher the molecular basis of the pathogenesis of human disorders and thus lead to novel or improved therapeutic strategies. We hope that the paradigms presented in this commentary of how diversification and regulation of PTMs in DUBs are achieved will guide future research.


The results of the [3H]uridine uptake study showed that the replacement of the naphthalene moiety with the benzene moiety could abolish the inhibitory effects on ENT1 and ENT2. The addition of chloride to the meta position of this benzene moiety could restore only the inhibitory effect on ENT1 but had no effect on ENT2. However, the addition of the methyl group to the meta position or the ethyl or oxymethyl group to the para position of this benzene moiety could regain the inhibitory activity on both ENT1 and ENT2. The presence of a halogen substitute, regardless of the position, in the fluorophenyl moiety next to the piperazine ring was essential for the inhibitory effects on ENT1 and ENT2. Among the analogues tested, compound 3c was the most potent inhibitor. Compound 3c reduced Vmax of [3H]uridine uptake in ENT1 and ENT2 without affecting Km. The inhibitory effect of compound 3c could not be washed out. Compound 3c did not affect cell viability, protein expression and internalization of ENT1 and ENT2. Therefore, similar to FPMINT, compound 3c was an irreversible and non-competitive inhibitor. Molecular docking analysis also showed that the binding site of compound 3c in ENT1 may be different from that of other conventional inhibitors. It is expected that structural modification may further improve its potency and selectivity and lead to the development of useful pharmacological agents.Equilibrative nucleoside transporters (ENTs) play a vital role in nucleotide synthesis, regulation of adenosine function and chemotherapy. Current inhibitors of ENTs are mostly ENT1-selective. Our previous study has demonstrated that 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N--1,3,5-triazin-2-amine (FPMINT) is a novel inhibitor of ENTs, which is more selective to ENT2 than to ENT1. The present study aimed to screen a series of FPMINT analogues and study their structure-activity relationship. Nucleoside transporter-deficient cells transfected with cloned human ENT1 and ENT2 were used as  FPMINT and its analogues were purchased from TopScience . Other chemicals were obtained from Sigma-Aldrich .Culture media and supplements were purchased from Thermo Fisher Scientific . Antibodies were obtained from Alomone Labs . Sulfo-NHS-SS-biotin was purchased from Thermo Fisher Scientific  uridine  for 1 min. Passive uridine uptake was determined by incubating the cells in HEPES-buffered Ringer’s solution containing [3H]uridine in the presence of 0.5 mM NBMPR. The cells were rapidly washed five times with ice-cold phosphate-buffered saline (PBS). After air-drying, the cells were solubilised by overnight incubation in 500 μl of 5% Triton-X100. Next, 300 μl of the cell lysates were mixed with 2 ml of scintillation liquid. The radioactivity of each sample was quantified by a scintillation counter. The protein content in each well was determined spectrophotometrically using a commercial bicinchoninic acid assay . Uptake values were expressed as picomoles of [3H]uridine per milligram of cellular protein per minute.Confluent monolayers of PK15NTD/ENT1 and PK15NTD/ENT2 cells in 24-well plates were washed thrice with HEPES-buffered Ringer’s solution containing (mM): 135 NaCl, 10 glucose, 5 KCl, 5 HEPES, 3.33 NaH3c or an equivalent volume of a vehicle (0.5% dimethyl sulfoxide (DMSO), as a control) in a serum-free medium for another 24 or 48 h. For measuring cell membrane integrity, the lactate dehydrogenase (LDH) released into the culture medium was measured with a detection kit  according to the manufacturer’s instructions. The absorbance at 490 nm was measured with a microplate absorbance reader. For measuring cell viability, 0.5 mg/ml methylthiazolyldiphenyl-tetrazolium bromide (MTT) was added to each well. The cells were further incubated for 4 h at 37°C to allow the formation of MTT formazan crystals. Finally, 100 μl of DMSO was added to lyse the cells and solubilise the crystals. The absorbance was measured at 560 nm, with the absorbance at 655 nm serving as the background.PK15NTD/ENT1 and PK15NTD/ENT2 cells were seeded onto 96-well plates until they reached 80–90% confluent. The cells were then treated with different concentrations of compound 3c or an equivalent volume of a vehicle (0.5% DMSO) for 24 h or 48 h. The cells were washed thrice with ice-cold PBS. Total protein was extracted by cell lysis in a lysis buffer  with a protease inhibitor cocktail . The protein content was measured by bicinchoninic acid assay. Protein samples were separated by 12.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% (w/v) non-fat dry milk in PBS for 1 h at room temperature and then incubated with rabbit polyclonal anti-ENT1 or anti-ENT2 primary antibodies  (1:200 [v/v] dilution in a blocking solution) at 4°C overnight. After washing thrice with PBS containing 0.02% (v/v) Triton X-100, the membranes were then incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies  at room temperature for 2 h. The membranes were then again washed, and the bound secondary antibodies were detected using an enhanced chemiluminescence kit . The antibodies recognized ENT1 as a protein of 40 kDa, and ENT2 as proteins of 50 and 47 kDa , but internalized transporters were protected and still contained biotin. Subsequently, 50 mM iodoacetamide in PBS was used to neutralize glutathione. Cells were then immediately solubilized in extraction buffer . After centrifugation at 13,000 x g for 5 min, supernatants with equal amounts of total proteins were agitated with streptavidin-agarose beads at 4°C overnight. Finally, the biotinylated transporters in the pull-down complexes, which should represent internalized transporters, were eluted by Laemmli buffer and resolved by SDS-PAGE as described above. Antibody against ENT1 or ENT2 was used to detect internalized transporters in the pull-down complexes and total transporter expressions in cell extracts.To label all surface proteins, after washing with ice-cold PBS, PK15NTD/ENT1 and PK15NTD/ENT2 cells were incubated with sulfo-NHS-SS-biotin (300 μg/ml) for 30 min at 4°C to biotinylate cell surface proteins. The unbound biotin was washed away by PBS containing 0.1% BSA at 4°C. The biotinylated cells were then incubated with the original growth media with or without 50 µM compound http://www.rcsb.org/pdb) (PDB ID: 6OB7). Two-dimensional structures of compound 3c and draflazine were downloaded from the PubChem database (http://pubchem.ncbi.nlm.nih.gov)  and was transformed into three-dimensional structure using Chem3D 19.0 software. The structures of ENT1, compound 3c and draflazine were pretreated and standardized with the Protein Preparation Wizard module of Schrödinger Maestro suite 2015 and the LigPreg module of Schrödinger LigPrep suite 2015, respectively. Autodock Vina software and Lamarckian Genetic Algorithm program were used to analyse the molecular interactions between ENT1 and drugs with default parameters. ENT2 was not studied because its crystal structure is not available.The X-ray crystal structure of human ENT1 was obtained from the Protein Data Bank . Data are expressed as mean ± standard error (SE) of three experiments performed in triplicate. The IC3H]uridine uptake in PK15NTD/ENT1 and PK15NTD/ENT2 cells were 18.42 ± 2.93 pmol/mg protein/min and 61.35 ± 6.67 pmol/mg protein/min, respectively. The dose-response curves for each FPMINT analogue against ENT1 and ENT2 are shown in 50 values for ENT1 and ENT2 were shown in PK15NTD/ENT1 and PK15NTD/ENT2 cells were used to screen a series of FPMINT analogues listed in 1a, 1b, 1c, 1d and 1e are the analogues of FPMINT, with modification in the N-naphthalene moiety. Compound 1a and 1e showed negligible effects on both ENT1 and ENT2. Compound 1b inhibited ENT1 with an IC50 value of 1.82 µM, but it had no effect on ENT2. Similar to FPMINT, compound 1c was more selective to ENT2 than to ENT1. The IC50 values of compound 1c against ENT1 and ENT2 were 171.11 µM and 36.82 µM, respectively. Although the potencies of compound 1c against ENT1 and ENT2 were lower than that of FPMINT, the selectivities of FPMINT and compound 1c were comparable . Unlike compound 1c, compound 1d was more selective to ENT1 than to ENT2 with IC50 values of 0.59 µM and 77.12 µM, respectively.Compounds 2a, 2b, 3a, 3b and 3c are the analogues of FPMINT, with changes in the N-naphthalene moiety and/or the fluorophenyl moiety. Compound 3a had no effect on both ENT1 and ENT2. Compounds 2a and 3b inhibited ENT1 with IC50 values of 104.92 µM and 1.65 µM, respectively. However, they did not inhibit ENT2. Compounds 2b and 3c inhibited both ENT1 and ENT2, but they were relatively more selective to ENT2. The IC50 values of compounds 2b and 3c against ENT1 were 12.68 µM and 2.38 µM, respectively, whereas their IC50 values against ENT2 were 2.95 µM and 0.57 µM, respectively. Compared to FPMINT, compound 3c showed a stronger potency, but compound 2b showed a weaker potency. However, their selectivities were close to that of FPMINT .Compounds 3c was selected for further investigation. The kinetic study of [3H]uridine transport was performed by measuring the concentration-dependent uptake of [3H]uridine. This study could provide clue about the mode of action of compound 3c. The uptake of [3H]uridine by ENT1 and ENT2 was saturable and conformed to Michaelis-Menten kinetics. Compound 3c decreased the Vmax of ENT1 but had no effect on Km. Specifically, the apparent Vmax values were 2236 ± 56.32, 2083 ± 36.80, 1867 ± 27.63, 1377 ± 37.81 and 732.9 ± 12.02 (pmol/mg protein/min) and the Km values were 0.143 ± 0.018, 0.142 ± 0.012, 0.143 ± 0.010, 0.144 ± 0.019 and 0.142 ± 0.011 (µM) in the presence of 0, 0.01, 0.1, 1 and 10 µM compound 3c, respectively  was 2.79 μM  and the Km values were 0.220 ± 0.035, 0.220 ± 0.041, 0.228 ± 0.059, 0.229 ± 0.050 and 0.226 ± 0.051 (µM) in the presence of 0, 0.01, 0.1, 1 and 10 µM compound 3c, respectively . The cells were then washed five times with buffer. After wash-out for every single time [3H]uridine uptake was measured. The inhibitory effects of compound 3c on both ENT1 and ENT2 were only partially reversed by washing  was much stronger than the analogue with an ethyl substitute . Moreover, the analogue with a chloride substitute at the meta position of this benzene ring  was slightly stronger than the analogue with a methyl substitute . The reason for this phenomenon is not known, but a common characteristic of the chloride and oxymethyl substitutes is that they possess lone pairs of electrons, which may increase the interaction with certain functional groups in the binding site of ENT1 through hydrogen bonds.First, the change from the naphthalene moiety to the benzene moiety abolished the effect on ENT1. This finding was confirmed based on the data of compounds 2a) reduced but not abolished the inhibitory effect on ENT1. Replacing the fluoride substitute at the ortho position with the chloride substitute at the meta position  did not significantly affect the inhibitory effect on ENT1. Moreover, comparison of the results of compounds 1b and 3b showed that changing the fluoride substitute from the ortho position to the para position did not alter the inhibitory effect on ENT1. It is apparent that the presence of a halogen substitute, regardless of its position on the phenyl moiety, was critical to the inhibitory effect on ENT1, probably through the electrostatic interaction with the binding site.Another determinant for the inhibitory effect on ENT1 is related to the fluorophenyl moiety next to the piperazine ring. The removal of the fluoride substitute from the fluorophenyl moiety  not only decreased but also completely eliminated the inhibitory effect on ENT2. Similar to that for ENT1, the change of the fluoride substitute at the ortho position to the chloride substitute at the meta position  restored the inhibitory effect on ENT2. Modification of the N-naphthalene moiety also greatly affected the inhibitory effect on ENT2. The replacement of the naphthalene moiety with the benzene moiety  not only abolished the inhibitory effect on ENT1 but also on ENT2. In contrast to that for ENT1, the addition to a chloride substitute to the meta position of the benzene ring could not restore the inhibitory effect on ENT2. This assumption was clarified when we compared compounds 1a against 1b, and compounds 3a against 3b. However, the addition of the ethyl or oxymethyl group to the para position of this benzene ring  enabled the compounds to regain the inhibitory activity, although the inhibitory effect was smaller than that of FPMINT. It remains unclear whether the presence of a para-substitute facilitates the binding of the compounds to ENT2. However, we believe that the methyl group in this benzene moiety may also be of paramount importance. The reason is that the change of the chloride substitute at the meta position of this benzene moiety  to the methyl substitute  could drastically make the compound, which was originally insensitive to ENT2, to become highly sensitive to ENT2.The presence of a halogen substitute on the fluorophenyl moiety played an even more critical role in the inhibitory effect on ENT2 because the absence of fluoride , which occupies the central cavity of ENT1 . Kinetic dilazep . Moreove dilazep . Interes of ENT1 , which i3c, they are not cytotoxic to PK15NTD cells. Nevertheless, further experiments are still required to completely reveal their safety profile. It is believed that the optimisation of the chemical structures of FPMINT and compound 3c may further improve the selectivity to ENT2. Development of a selective ENT2 inhibitor will be a useful research tool or even a potential therapeutic agent. ENT2 is particularly abundant in skeletal muscle and adrenal gland (It has been reported that certain piperazine derivatives may have cytotoxic effects on hepatocytes and cancer cell lines . Althougal gland  but its al gland . Moreoveal gland . These eIn conclusion, our study has provided evidence that the modification of the N-naphthalene moiety and the fluorophenyl moiety in FPMINT can change the inhibitory potency and selectivity for ENT1 and ENT2. The insights obtained from the structure-activity relationship may be useful in designing and developing more potent and selective ENT2 inhibitors.


Phosphorylation, which is mediated by protein kinases and opposed by protein phosphatases, is an important post-translational modification that regulates many cellular processes, including cellular metabolism, cell migration, and cell division. Due to its essential role in cellular physiology, a great deal of attention has been devoted to identifying sites of phosphorylation on cellular proteins and understanding how modification of these sites affects their cellular functions. This has led to the development of several computational methods designed to predict sites of phosphorylation based on a protein’s primary amino acid sequence. In contrast, much less attention has been paid to dephosphorylation and its role in regulating the phosphorylation status of proteins inside cells. Indeed, to date, dephosphorylation site prediction tools have been restricted to a few tyrosine phosphatases. To fill this knowledge gap, we have employed a transfer learning strategy to develop a deep learning-based model to predict sites that are likely to be dephosphorylated. Based on independent test results, our model, which we termed DTL-DephosSite, achieved efficiency scores for phosphoserine/phosphothreonine residues of 84%, 84% and 0.68 with respect to sensitivity (SN), specificity (SP) and Matthew’s correlation coefficient (MCC). Similarly, DTL-DephosSite exhibited efficiency scores of 75%, 88% and 0.64 for phosphotyrosine residues with respect to SN, SP, and MCC. Protein phosphorylation is an important posttranslational modification (PTM) that regulates many cellular activities and contributes to the etiology and progression of several pervasive diseases, including cancer, diabetes, cardiovascular disease, and neurodegeneration. In eukaryotic cells, phosphorylation, and subsequent dephosphorylation, occurs on serine (S), threonine (T), and tyrosine (Y) residues located on the protein surface. To date, more than two-thirds of the ∼21,000 proteins encoded by the human genome have been shown to be phosphorylated, making phosphorylation one of the most wide-spread and broadly studied protein PTMs . The prePhosphorylation site prediction has recently emerged as an important problem in the field of bioinformatics. As a result, many phosphorylation site prediction tools have been developed to predict both general and kinase-specific phosphorylation sites . For insWhile many phosphorylation site prediction tools have been developed over the past decade to identify putative sites of S, T, and Y phosphorylation , computaOne of the primary reasons for the proliferation of phosphorylation site predictors over the past decade is the availability of large databases cataloging experimentally identified phosphorylation sites, such as PhosphoSitePlus and PhosphoELM . UnfortuHere we have developed the first general phosphatase site prediction tool. Unlike phosphatase-specific methods, which are designed to predict both the site of dephosphorylation and the phosphatase mediating the dephosphorylation event, our general dephosphorylation site prediction method is able to identify putative sites of dephosphorylation irrespective of the phosphatase mediating the dephosphorylation event. This is analogous to the results obtained by MS/MS-based experiments, where information about the responsible phosphatase is not known. Importantly, phosphatase-specific methods are currently restricted to predictions for only three phosphatases , which represent a very small fraction of phosphatases encoded by the human genome. This is likely due, in part, to limited information about the specific phosphatase that mediates a given dephosphorylation event. Therefore, general dephosphorylation site prediction methods offer distinct advantages when the primary goal is to predict whether or not a given site is dephosphorylated.The human DEPhOsphorylation Database, DEPOD, is a database of dephosphorylation sites that was recently expanded in an updated version in 2019 . DEPOD aOnce constructed, the dataset was further divided into training and test sets, such that 80% of the data was used to train the models and the remaining 20% of the data was kept aside for independent testing. This training-test dataset, which we termed the DEPOD-19 dataset , consistThough phosphorylation is one of the most wide-spread and well-studied PTMs in eukaryotes, comprehensive lists of dephosphorylation sites are scarce. This is likely due to the lack of computational studies in the field and technical challenges associated with the detection of dephosphorylation sites. Therefore, in order to enlarge the dephosphorylation site dataset , we did During sequence extraction, a sub-sequence with window size of 33 centered around the site of interest was created in a manner similar to that described for the DEPOD-19 dataset above. Long Short-Term Memory (LSTM) models are known to provide good performance with sequence data . LSTM ust) consists of a sigmoid layer and a tanh layer. The sigmoid layer filters the previous state to select the relevant cell states for the context while the tanh layer provides a range of values to take to the selected states. The forget layer (rt) consists of a sigmoid layer, which filters the irrelevant previous cell states by dropping them out. The output layer (hþt) employs a tanh layer to provide an update to the selected states, as provided by the input layer , which uses twice the number of neurons as a conventional LSTM layer. The double neurons create two sets of networks, moving in both the forward and the reverse directions . Thus, aPTSPSYSPTSPSYSPSNYTPTSPNYSPTSPSYS (red) in the center represents the target residue, the forward LSTM network would predict the probability of having S, given the knowledge of the residues preceding it  while the backward/reverse LSTM network would predict the probability of having S, given the knowledge of residues following it . The window sequences were integer encoded, such that each character in the sequence was replaced by its corresponding integer value. The integer encoded sequences were then fed to the embedding layer, which provides an embedding dimension of 21, which is known to be optimal based on our previous studies  are different. Moreover, the extensive study of phosphorylation sites has resulted in a comparatively large dataset of phosphosites, while the amount of information about dephosphorylation events has led to a relatively sparse dataset. Taken together, these observations suggest that a transfer learning strategy could be applied to dephosphorylation site prediction.Recently, deep learning has been used to solve various problems in bioinformatics . One of Previous studies suggest that, when trained on images, deep learning networks tend to learn first-layer features that do not appear to be specific to a particular task . Such fiWith the exception of a handful of dual specificity kinases and phosphatases, most kinases and phosphatases recognize either S/T or Y residues. Therefore, as is common in phosphorylation site prediction, we considered two models: one for S and T residues and another for Y residues. Thus, distinct phosphorylation and dephosphorylation datasets were formed and designated the Phos-ST and Phos-Y datasets and the Dephos-ST and Dephos-Y datasets.During transfer learning, three important questions need to be answered: (a) what to transfer, (b) when to transfer, and (c) how to transfer. Therefore, to allow our framework to accommodate smaller datasets, we applied a two-step transfer learning scheme that included a pre-training step and a fine-tuning step . The preThe pre-training step involves the training of our Bi-LSTM model  on the available phosphorylation data , which aSimilarly, for the prediction of Y dephosphorylation sites, we experimented with performing transfer learning from Phos-ST-to-Dephos-Y as well as Phos-Y-to-Dephos-Y. These studies suggested that the Dephos-ST-to-Dephos-Y transfer worked the best. Thus, the pre-training step involved training the Dephos-ST model, initialized with transfer-learned weights from Phos-ST on the Dephos-ST dataset. During the fine-tuning step, we retrained all layers on the Dephos-Y dataset. Though varying the layers that were kept frozen or re-trained had less impact in performance, retraining all layers helped in attaining more consistent results.Finally, we also employed the transfer learned Dephos-Y model on the available phosphatase specific datasets  for PTP1To evaluate the performance of each model, we used a confusion matrix to determine Sensitivity (SN), Specificity (SP), Accuracy (ACC) and the Receiver Operating Characteristic (ROC) curve as the performance metrics. The models were evaluated using five-fold cross-validation on the benchmark training dataset and an independent test set.ACC describes the correctly predicted residues out of the total residues (Eq. 1). Meanwhile, SN defines the model’s ability to distinguish positive residues (Eq. 2) and SP measures the model’s ability to correctly identify the negative residues (Eq. 3). Matthews Correlation Coefficient (MCC) is the calculated score that takes into account the model’s predictive capability with respect to both positive and negative residues (Eq. 4). Likewise, the ROC curve provides a graphical representation of the diagnostic ability of the classifier. The area under the ROC curve (AUC) is used to compare various models, with the models having the highest AUC scores generally performing better in classification than those with lower AUC scores.To efficiently identify sites that are likely to be dephosphorylated in proteins, we sought to develop a dephosphorylation site prediction tool using the recently expanded DEPOD-19 dataset . To thisThough the DEPOD-19 dataset has recently been expanded to include 584 total sites, it still represents a relatively small dataset for model development using machine learning strategies. Therefore, to further expand the dataset, we conducted a comprehensive literature search for dephosphorylation sites. This yielded an additional 1,898 sites whose phosphorylation status decreased within an hour of treatment in mitotically arrested cells . We thenThe observed gains are likely due to an increase in the size of the dataset, consistent with several reports that suggest that deep learning models perform well on large datasets and that an increase in the size of the dataset can increase the performance of the resulting model . HoweverIn contrast to dephosphorylation sites, phosphorylation sites have been extensively annotated, totaling 484,110 sites in 20,217 proteins  yielded models that performed well in most metrics, but not as well as the window size 31 Y dephoshorylation model generated using DTL-DephosSite-ST as the source model and the ComDephos dataset as the target dataset . For insHere, we describe a strategy that combines deep learning with transfer learning to develop general dephosphorylation site predictors of S/T and Y residues. To our knowledge, the resulting models, termed DTL-DephosSite-ST and DTL-DephosSite-Y, are the first general dephosphorylation site predictors for S/T and Y dephosphorylation, respectively. Deep learning-based models have recently been developed for several important PTMs, including phosphorylation, methylation, acetylation, and succinylation, to name a few . Similarhttps://github.com/dukkakc/DTLDephos to further contribute toward the study of dephosphorylation.During this project, we also expanded the DEPOD-19 dephosphorylation dataset 3.25-fold to create computational datasets of dephosphorylation. Importantly, this study relies upon the correlation between the cellular processes of phosphorylation and dephosphorylation. We have attempted to measure the level of transferability between phosphorylation and dephosphorylation. Similar correlations are also likely to be found for other PTMs where the forward and reverse reactions are catalyzed by different classes of enzymes, such as methylation/demethylation and acetylation/deacetylation. Prediction of sites of these modifications may thus be amenable to transfer learning. Likewise, PTMs that differ in the molecular characteristics of the PTM itself, but which utilize related enzymes, such as ubiquitin E3 ligases and SUMO E3 ligases, may also be amenable to transfer learning. Finally, all datasets and code developed during this study has been made freely available to the bioinformatics community at https://github.com/dukkakc/DTLDephos.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: DK, RN, MK, and DC conceived, designed the experiments, and revised the manuscript. MC, HI, NT, and SC performed the experiments and data analysis. MC, DK, RN, and SC wrote the manuscript. All authors read and approved the final manuscript.MK is a paid consultant for Orion Pharma. The remaining 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.


Scanning electron microscopy (SEM) is a powerful imaging technique able to obtain astonishing images of the micro- and the nano-world. Unfortunately, the technique has been limited to vacuum conditions for many years. In the last decades, the ability to introduce water vapor into the SEM chamber and still collect the electrons by the detector, combined with the temperature control of the sample, has enabled the study of ice at nanoscale. Astounding images of hexagonal ice crystals suddenly became real. Since these first images were produced, several studies have been focusing their interest on using SEM to study ice nucleation, morphology, thaw, etc. In this paper, we want to review the different investigations devoted to this goal that have been conducted in recent years in the literature and the kind of information, beyond images, that was obtained. We focus our attention on studies trying to clarify the mechanisms of ice nucleation and those devoted to the study of ice dynamics. We also discuss these findings to elucidate the present and future of SEM applied to this field. Ice nucleation and water freezing are naturally occurring processes on Earth with an enormous impact on climate, geology, and life. Understanding the mechanisms governing these processes is essential for predicting the future of our planet. Without knowing more about ice formation, it is impossible to build robust snow or ice-cloud theories for atmospheric and climate models or to extrapolate laboratory studies to environmental conditions with confidence. A few years ago, the ten questions that science needs to examine to gain this knowledge were established . The firThe formation of ice in the troposphere, both on the Earth’s surface and in clouds, usually takes place on solid surfaces through heterogeneous nucleation. Many factors can affect heterogeneous nucleation and not all surfaces induce ice nucleation with the same efficiency. The properties of surfaces at the nanometer range play a key role in heterogeneous nucleation due to the size of the initial ice nuclei, which is in the order of few to tens of nanometers. To understand how that happens, the molecular bases of the interaction of water molecules with surfaces need to be studied. Many different techniques can be used to study water interaction with surfaces and heterogeneous nucleation, such as photon spectroscopy , opticalIn recent years, with the rapid advancement in instrumentation as well as simulation capabilities, important advances in the process of understanding of ice nucleation and ice dynamics emerged ,10,11,12Environmental scanning electron microscopy (ESEM) was developed to overcome the limitations of the classical SEM, operated at high vacuum condition, required for the operation of the electron gun. This condition limited the range of specimens compatible with the technique to conductive, dry, and stable samples. The development of differentially pumped diaphragms allowed the introduction of a gas medium in the specimen chamber while maintaining high vacuum conditions for the electron gun ,15,16. TThe first commercial ESEM was presented in 1980 by the Electroscan Corporation, but, soon after, other companies followed to introduce the ESEM capabilities into their instruments. Nowadays, ESEM chambers are available from different manufactures and are widely used in investigation in many areas of science, such as materials sciences, biology, or nanotechnology. Before ESEM, ice nucleation investigation was based on diverse techniques developed for that purpose including single particle methods, such as rapid expansion cloud chamber , mixing The technique is continuously evolving and, as a recent example, a new system combining ESEM capabilities with an X-ray spectroscopy analytical platform was developed to directly observe the evolution of organic particles with increasing RH . The newDifferent mechanisms of heterogeneous ice formation in the troposphere have been proposed : contactIn 2007, Frank Zimmermann et al.  presenteMore evidence on the dependence on mineralogy was presented by the same authors the following year. Using the same technique, they examined ice nucleation on nine pure minerals present in mixed-phase clouds . The autA novel ice nucleation ESEM experimental platform with a cryogenic temperature controller was presented in 2016 by Wang et al. . The aimA breakthrough in the understanding of the nature of ice growth on mineral samples came in 2017, when Kiselev et al.  directlyThe ESEM higher resolution capabilities were also key in exposing the microscopic step by step pathway of ice crystals’ formation in a supersaturated water vapor environment where two different origins of the steps were unveiled . The hexIce nucleation on porous particles can be described by the pore condensation and freezing theory, which was revisited and updated in 2019, based on the latest experimental results . In thosIn our study, we were able to observe, in situ and in real-time, the filling of the pore and the emerging out of it in the form of a hexagonal ice crystal a. Consec2 nanoparticles uniformly distributed across rGO structure was tested as an ice nucleating promotor. It was found that the composite promotes hexagonal ice growth via a lattice match between the ice and its structure. The in situ ESEM ice growth observation confirmed the enhanced ice nucleation performance by ice crystal formation events starting from −8 °C and 5–8% RH. In addition, it was observed that ice nucleation would start in large cavities in the rougher regions of the composite, in line with other, previously mentioned studies.Carbon-based particles found in the troposphere account for up to 50% of all the particles, many of them being of graphitic-like structure, called soot particles. For this reason, carbon nanomaterials are studied as ice nucleation promoters, especially graphene and its derivatives due to their intrinsic and functionalized surface properties ,68,74,75Highly oriented pyrolytic graphite (HOPG) is another representative member of a soot particles’ family and one of the components of dust present in the atmosphere. Hence, it was chosen as a model ice nucleator in another study involving ESEM . The resAlthough many works focus on pristine minerals for the studies of ice formation, in real atmospheric conditions, soil-derived matter composed of a mixture of mineral and organic particles contribute to the ice nucleation process. Hence, important aspects when studying cloud formation, precipitation, and soil-derived nutrient cycling are the interactions between mineral particles, biological matter and ice ,79. Ice Conocephalum salebrosum and Marchanthia polymorpha L. subsp. ruderalis in 2021 [Water freezing and ice formation are important from the point of view of climate, but also affect the survival of plants. The survival of some plants at temperatures below the freezing of water is conditioned by dehydration mechanisms through extracellular ice formation. This mechanism was visualized by ESEM on vesicular plants, such as  in 2021 . Ice forIn the initial modern studies on water freezing, around 1930s, important discoveries on ice formation were claimed based on observation by cameras and optical microscopes. The old quote that claimed that there are no two identical snowflakes  was confWith the advancement in resolution capabilities of the ESEM technique, high magnification images of ice crystals were possible to be obtained. In a study that compares hexagonal ice crystals grown in situ in the ESEM with ice crystals grown ex situ in a diffusion chamber , the mesVery recently, the complete process of ice growth and sublimation on surfaces was followed by in situ ESEM, from the initial ice growth through the coalescence of the crystals and formation of polycrystalline film to the sublimation of ice on an oxidized Si wafer . The fin+, Cl−, and Br−, and have been observed in snow, ice cores, and aerosols forming a large chemical reservoir [During the process of water freezing, the impurities present in it are trapped inside to become a part of the structure of the formed ice and/or snow. These impurities are stored, transformed, and eventually released from the crystallized water. The information about the location and speciation of this contamination under varying environmental conditions is critical for assessing their reactivity and fate ,96,97. Teservoir ,100,101.eservoir . Answerseservoir . The briFrom an industrial point of view, ice formation and accretion can present a serious problem causing severe accidents in certain areas, such as transportation, power networks, infrastructures, aviation, wind turbines, among others. Hence its prevention is of high importance ,105,106.Using SHB surfaces is not the only approach for anti-icing. Another approach consists of creating hydrophilic polymer coatings ,114, whiAs previously mentioned, the electron beam interaction with the specimen under observation may play a role in some, if not all the studies conducted under ESEM. However, even if counter-intuitive, this effect may be used to the advantage of a study. The most recent example is the study of the growth of amorphous solid water (ASW) on the electrically charged surface of sapphire, used as a source to charge the electrons coming out from the gun of the scanning electron microscope . After tThe playwright and theatre director Henrik Ibsen first said, “A thousand words leave not the same deep impression as does a single deed”. After his death in 1906, this quote was rephrased into what we know now, “A picture is worth a thousand words”, which is now a common saying. This explains very well the value that the ESEM technique has in the field of the understanding of heterogeneous ice nucleation. It has helped to visualize and to better understand the processes governing water transitions from gas, to liquid, to solid, and vice versa. The ice nucleation, growth and sublimation on particles, surfaces and organic matter were visualized in the ESEM and characterized spectroscopically by many scientists providing relevant information on the subject. We believe one of the main advantages of ESEM is the ability to determine with nanometric resolution the sites where ice nucleation takes place and even visualize the initial steps of ice nucleation, with crystals in the order of only hundreds of nm. This lateral resolution combined with the possibility of chemical analysis of the surface with the same resolution opens a wide field to investigate the main question about ice nucleation still with more shadows than light; what makes a site on a surface or particle so special to induce ice nucleation more efficiently than another sites. So far, the experiments shown in this paper mainly explored ice nucleation on “natural” materials, such as feldspars, silica, graphite, and plants. In this paper, by “natural” we mean materials not designed with specific properties or functionalities. However, few examples of designed surfaces have shown some interesting properties towards water and ice, such as the hydrophilic islands on superhydrophobic surface. We believe with the capabilities of nanotechnology and ESEM resolution, more of these kinds of special surfaces can be designed and fabricated with sites of well-defined properties as models to determine how different surface properties interplay to induce ice nucleation. The same can be applied for surfaces designed to control ice dynamics spatially, as observed in


This study proposed a method for fabricating 3D microstructures of ice without a supporting material. The inkjet printing process was performed in a low humidity environment to precisely control the growth direction of the ice crystals. In the printing process, water droplets (volume = hundreds of picoliters) were deposited onto the previously formed ice structure, after which they immediately froze. Different 3D structures (maximum height = 2000 µm) could be formed by controlling the substrate temperature, ejection frequency and droplet size. The growth direction was dependent on the landing point of the droplet on the previously formed ice structure; thus, 3D structures could be created with high degrees of freedom. Using inkjet printing technology, a method has been developed for fabricating 3-D ice microstructures, where the ice patterns possess geometries with a resolution of tens of micrometers. Recently, inkjet technology has gained attention for use in micromanufacturing, with different deposition processes being utilized to apply a material onto a substrate. Through the phase transition of water to ice, water offers many advantages as a material in this regard, though existing technologies demand expensive equipment and a supporting layer. A team headed by Zhihong Li at Peking University, China has succeeded in developing a method where inkjet printing is used to precisely control the growth of ice crystals in the fabrication process without needing a supporting layer. The authors believe their system offers considerable potential for application with biomicrofluidic devices, such as drug delivery systems. In micromanufacturing, inkjet deposition processes are used to produce a pattern of material on a substrate. Most micromanufacturing applications of inkjet technology deposit phase change liquid inks onto nonporous substrates, which solidify quickly after deposition. Examples of phase change materials in micromanufacturing applications include solders for electronic manufacturing4 and thermoplastics for free-form fabrication6. The control of spreading by solidification is a beneficial aspect of phase change materials for applications where the goal is to limit spreading and obtain the smallest spot for a given droplet size7.In the past decade, inkjet technology has become recognized in the technical community as a highly capable tool for manufacturing, particularly micromanufacturing8. Studies have been applied to determine the macroscale mechanism and process of icing, such as freezing front propagation10, supercooling phenomenon12, ice spike phenomenon13, ice layer spreading along a substrate14 and freezing of droplets16 and bubbles17. Ice has also been widely used in microfabrication, such as ice templates18 for biomaterial systems, ice lithography21 for nanopatterning methods and ice blocks for microfluidic channels created by laser processing22. However, these technologies are based on removal techniques and require expensive equipment and an additional supporting layer or protection layer.Water is the most common substance in nature and has substantial advantages in free contamination. The phase transition from water to ice is a phenomenon that often occurs23. A small liquid droplet can be printed separately from the nozzle onto a cold surface, after which the liquid droplet immediately freezes into an ice crystal. Then, an increasing number of water droplets can be printed and interconnected to create a specific structural design. This ice structure can then be used in various applications under the ice point. However, in its current form, this ice printing method is limited to simple planar structures close to the substrate, such as microcapsule ice arrays for reagent presealing25 and parallel ice channels for microfluidics26. In this paper, we optimized the process parameters to precisely control the growth direction of ice crystals, fully realizing the concept of 3D ice printing. A variety of ice structures with different geometries, sizes, heights and overhanging structures were successfully produced. In contrast to other 3D printing methods, such as fused deposition modeling and stereolithography, the ice printing method does not need additional supporting structures or removal processes, which simplifies the geometric design and avoids the unnecessary introduction of other chemicals. Moreover, the 3D ice printing method does not require specific substrates. The ice structure can be formed on any theoretical substrate that is sufficiently cold for icing, such as glass, metal, silicon, and polymer films. Furthermore, 3D-printed ice structures can be used as soft lithography molds for complex microfluidic channels, which is difficult or even infeasible for conventional photolithography methods. In addition, 3D ice printing has the potential to create 3D porous scaffolds of metal salt nanoparticles, which precipitate from the water during the freezing process and then occupy the spaces between the ice grains.Utilizing water as a phase change material in 3D printing, we proposed an economical additive manufacturing method to fabricate ice structures by inkjet printing, which is called “ice printing”A customized inkjet printing system has been made to demonstrate the 3D ice printing method. Figure The ice printing system can directly print 3D ice structures with micron resolution by additive manufacturing. The ice structures are flexible with respect to size, geometry and height. Figure The icing process is mainly affected by the type of solution, the temperature field above the substrate, the environmental humidity and the printing frequency.Glass substrates are used in the following discussion. The height of the printed features had a variation of 1–3.5%, which indicates the reproducibility of the ejected droplet volume. Therefore, inkjet printing was found to be a suitable patterning technique for highly reproducible processes.27. The supercooling phenomenon only occurs when droplets contact a clean substrate. When the droplets contact the printed ice structure, they always freeze. The supercooling phenomenon is a probabilistic event in the ice printing process because the droplets have a certain initial velocity when they hit the substrate, and the nonsteady state will help the droplets freeze. To reduce the probability of the supercooling phenomenon, an effective method is to minimize the temperature of the substrate. Related studies show that the crystallization rate of water reaches a maximum at approximately −48.15 °C, which implies the lower limit of metastability of liquid water28. Thanks to the two layers of Peltier coolers in the ice printing system, the current ice printing system can reach −56 °C. Another method is to modify the surface of the substrate, either introducing irregular structures to disrupt the steady state of the droplet or introducing particles to help form the ice core. For water containing impurities, its freezing point will change, which can be calculated using the following formulaThe formation of ice crystals is mainly influenced by the type of solution. Here, we divide the solutions into two categories. For ultrapure water, the supercooling phenomenon severely interferes with the formation of ice structures on cold substrates. As a small volume droplet is free of impurities and bubbles, even if the temperature is below 0 °C, it will remain liquid instead of freezingThe growth process of the ice crystal structure is mainly influenced by the temperature field around the substrate. Experiments have shown that ice structures should be produced at a maximum temperature of −4 °C; otherwise, the droplet freezes too slowly, destroying the intended structural design. The −4 °C isotherm height is the first reason limiting the height of the ice structure. Compared to the isolation container housing the entire printing system, the cooling system was very small. Therefore, we built a heat transfer model in an environment with an unlimited nitrogen supply. The temperature field above the substrate simulated by COMSOL Multiphysics is presented in Fig. d. The minimum value of d that still allowed the droplet to freeze stably was defined as dmin. As the ice height increases, the temperature of the ice surface may also increase. To reduce the thermal effect from the nozzle, the nozzle should be far from the ice, which means that dmin increases as the height of the ice structure increases. Second, the nozzle should not be too far from the ice structure. Because of the temperature gradient, the convection from the air is strong above the substrate, and a long distance will cause the flying droplet to have a random motion and deviate from the intended position. Considering the accuracy of the deposition on the substrate, a shorter distance between the nozzle and substrate will subsequently reduce the disturbance of the airflow on the flying droplet. The maximum value of d that still allowed the droplet to fly stably was defined as dmax. Figure dmax decreased as the height of the ice structure increased. For a stable fabrication process, d should always be within the bounds of dmin and dmax. The available intervals of d for ice structures with different heights is the second reason limiting the height of the ice structure. In accordance with the simulation results and previous experimental experience, when the height of the ice structure was less than 2 mm, the distance from the nozzle to the top of the ice structure was set to 1 mm.In addition, the nozzle also influenced the temperature field around itself, as shown in Fig. The preservation of an ice crystal structure is mainly influenced by humidity. In a stable low-temperature environment, the main factor affecting the stability of ice is sublimation, especially for microstructures fabricated by ice printing. The sublimation rate increases substantially with decreasing humidity. However, a dry atmosphere is necessary to avoid frosting on the cold surface, which will keep the substrate clean and the surface of the ice structure smooth. Otherwise, the water molecules in the environment will form ice crystals on their surfaces. Usually, the humidity is set below 0.01% RH. To control humidity, the whole system was placed inside a container to keep it isolated from the outside. Pure nitrogen, controlled by a flow meter, was passed into the container to adjust the humidity. Although evaporation and sublimation always exist in the printing and freezing process, this setup ensured that the water molecules diffusing into the environment had less effect on humidity. In the case of continuous printing, an ultralow humidity environment can exist for at least four hours.x-y plane. Hence, all of the droplets fell on the same point. The substrate was a glass slide, and the temperature of the substrate was −27 °C.The morphology of a frozen droplet is greatly affected by the printing frequency. In the discussion in this section, the nozzle did not exhibit displacement in the 29. Each droplet was a smooth sphere in the liquid state. When the droplet froze, because the freezing process occurred from the bottom up30 and the water swelled during the freezing process, the ice bead had a protrusion on the top. Although this phenomenon was inevitable, the angle of the protrusion was not affected by the rate of solidification but depended on the water density-to-ice density ratio31, the wetting angle32 and the substrate temperature33. For droplets of the same volume, the ice beads were always conical, so this phenomenon did not affect the resolution of ice printing technology but only limited the smoothness of microscale ice structures.When the printing frequency was lower than 20 Hz, the freezing of each droplet was independent. As the volume of the droplet was only hundreds of picoliters, the droplet had no impact or retraction on the substrate, and it froze in less than 50 ms, which was the main difference from the macroscale behavior of the dropletWhen the printing frequency was higher than 20 Hz and less than ~1250 Hz, the printing created torch-shaped ice structures with large tops and small bases, which was influenced by the time of the continuous printing . Different from the continuous printing mode, in the trigger mode, the number of droplets N instead of the printing time T is set for each printing action. When N is far less than f, it seems that the printing action only produces one “large droplet” on the previous ice structure. Under this circumstance, the printing action is called a trigger. This result allows a dynamic scaling of the ice printing technique, where the size of droplets in each trigger is adjusted according to the desired feature size: depending on the size of the nozzle (20–80 μm) and the number of droplets in each trigger (1–20), we could produce voxels from 30 to 200 μm in width.Furthermore, we observed faster vertical growth in the “trigger” mode. The trigger mode is a manual operation mode in which the printing frequency The growth properties of the ice crystals deposited on an ice structure were recorded using a CMOS camera with a strobe light  camera. When printing each droplet, we managed to make the droplets, which flew obliquely downward, contact the side of the ice structure. In this way, the droplet will continue moving downward along the surface of the ice structure instead of staying steadily on the top of the ice structure. Because the volume of the droplet is very small, it will freeze and become an extension of the existing ice structure before falling off of the ice structure. In this way, horizontal growth and diagonal downward growth of the ice structure are achieved. Figure x-y positioning capabilities of the movement platform  connected with a computer.A custom inkjet printing system was constructed to demonstrate the 3D ice printing method. Figure A cold surface was required for the working platform on which freezing occurs. The cooling system (which could reach a minimum temperature of −56 °C) consisted of two Peltier coolers and a radiating plate with circulating water at 5 °C. The DC voltage source controlled the heat transfer from the cold face to the hot face of the Peltier coolers. The radiating plate, which was connected to a water-cooling system, was used to release the heat from the hot side of the Peltier coolers. These two parts had a uniform layer of silver-containing thermal grease between them to enhance heat transfer. A platinum thermocouple was used to measure the working temperature of the cold surface in the cooling system. All the self-built cooling systems were surrounded by foam insulation to isolate the heat exchange with the environment.During the printing process, the droplets collided with the ice structure and then spread out and immediately froze via heat conduction without a rebounding motion. With a certain nozzle size and voltage signal, frozen layers were repeatedly formed with a constant thickness and diameter, leaving ice patterns on the substrate. Controlled by a computer, the modified inkjet printer can print droplets of different sizes at different frequencies in the desired location during the manufacturing process.The whole ice printing system was placed in a container with a nitrogen atmosphere to isolate the system from the outer environment. Icing is a complex physical process during which humidity and temperature are the main factors. Hence, a digital thermometer and hygrometer  were used to monitor the environment inside the container.The system is flexible, enabling the user to design custom deposition processes. For example, here, we added two CCD cameras (front and side) to enable visualization of the nozzle loading, the generated droplets and the printed structures.tsp file. The file was loaded into the printer software. The.tsp files were generated by a design assistant called TSAPS, which was provided by Shenteng Technology, China. Alternatively, the files can be generated by any third-party software capable of exporting CAD files.When manually controlled, the printing process was monitored by the two CCD cameras. The landing point of the droplet can be confirmed by observing the position on the screen. When the printing process was automated by a preset program, the trajectory, the movement speed and the voltage control of the nozzle were specified in a.Video 1Video 2Video 3Video 4Supporting Information File