Backward walking offers a unique challenge to balance and ambulation. This study investigated the characteristics of spatiotemporal gait factors and ankle kinematics during backward walking in people with chronic ankle instability. Sixteen subjects with chronic ankle instability and 16 able-bodied controls walked on a treadmill at their self-selected speed under backward and forward walking conditions. Gait speed, cadence, double limb support percentage, stride time variability, and three-dimensional ankle kinematics were compared between groups and conditions. During backward walking, both groups had significantly slower gait speed, lower cadence, and greater stride time variability. In addition, under backward walking condition, subjects in both groups demonstrated significant sagittal and frontal kinematic alternations, such as greater dorsiflexion and inversion following initial contact . However, there were no significant differences between groups in any of the measured outcomes. This indicates that subjects with chronic ankle instability adapt to self-selected speed backward walking similarly to healthy controls. Assessments with more challenging tasks, such as backward walking with dual task and backward walking at fast speed, may be more appropriate for testing gait impairments related to chronic ankle instability. CAI is characterized by repetitive episodes and subjective feeling of ankle ‘giving way’, and symptoms such as pain, swelling and limited motion4. Compared to healthy controls, individuals with CAI report quality-of-life deficits and functional limitations in addition to the physical impairments6.Chronic ankle instability (CAI) may be present in up to 40% of individuals who have previously experienced lateral ankle sprain2, it can occur even when the mechanical constraints at the ankle are intact7. Recent evidence suggest that CAI can be explained by sensorimotor deficits8. Arthrogenic neuromuscular inhibition10, ankle muscle weakness11, reduced ankle range of motion9, impaired sense of joint position12, and postural control are found in CAI.While mechanical factors, such as ankle ligaments hyperlaxity, may be responsible for CAI in some patients13. During walking, subjects with CAI may exhibit typical kinematic patterns of increased ankle inversion and a laterally deviated center of pressure throughout the stance phase of gait6. Conversely, Chinn et al.5 reported that CAI subjects demonstrated more inversion while jogging but not while walking.Altered movement patterns during functional tasks, including walking, are often described in individuals with CAI14. In addition, differences were also found in the coordinated movement of segments of the lower extremity. For example, during walking, patients with CAI demonstrated less variability in frontal-plane ankle-hip coupling and greater variability in ankle frontal-knee sagittal-plane motions than patients without CAI15. Challenging conditions, such as rapid movements, fast walking, and walking with a cognitive dual task, emphasize differences in movement patterns between subjects with CAI and healthy individuals16. This indicates that higher attentional demands during a functional task may further limit the ability of this population to control movement.Linear variability measures that investigated the amplitude of variability, such as coefficient of variation, and non-linear variability approaches that evaluated the dynamic aspects of variability using mathematical tools related to chaos theory, have both reported differences in gait variability between individuals with and without CAI. For example, individuals with CAI have increased variability in location of center of pressure during the initial stages of stance phase18. BW differs from FW in many aspects. Step length is shorter and gait speed is slightly slower during BW19. Kinematic and kinetic studies demonstrated greater range of ankle dorsiflexion, reduced plantar flexion, and more even plantar pressure distribution 21. BW requires greater muscle activity and has higher metabolic costs22. Moreover, BW involves increased activation of the sensorimotor control system due to altered or absent visual feedback23. BW may be extremely novel even for healthy individuals. Kurz et al.24 investigated gait variability and cortical activation in healthy adults during FW and BW and reported increase in sensorimotor cortical activation measured by functional near infrared spectroscopy and a greater stride-time variability during BW. It has also been reported that exercise of BW in untrained healthy adults caused neural adaptations25. Overall, BW offer a unique challenge to balance and movement. Thus, BW is used increasingly in rehabilitation programs to promote balance control17.Backward walking (BW) is an activity with additional complexity compared to regular forward walking (FW). Although some studies have shown that BW is a mirror image of FW, considering that initial contact during BW is done by the toe instead of the heelDue to the nature of a relatively untrained and challenging gait task, assessing characteristics of BW among people with CAI, as compared to healthy controls, may provide additional information regarding sensorimotor control in this population. Furthermore, the ability to walk backward may be a useful measure of mobility, as well as balance training strategy. Thus, it is important to quantify the performance of BW in people with CAI.Therefore, this study investigated the characteristics of spatiotemporal gait factors and ankle kinematics during BW among people with CAI. For this purpose, we compared changes between FW and BW among individuals with and without CAI. We expected that only BW would differ between these populations. Specifically, we hypothesized that during BW, individuals with CAI would have slower gait speed and increased ankle inversion, as compared to healthy controls.26. To detect a true and meaningful difference of 0.1 m/s in BW gait speed, with standard deviation of ± 0.1, power of 80%, and a 95% confidence level, a sample of 16 subjects in each group was needed. Consequently, 16 subjects with CAI and 16 healthy controls participated in the study. The enrollment criteria for the CAI group were based on previously established standards to identify individuals with CAI27. Participants with CAI were included if they met the following criteria: (i) history of at least one significant ankle sprain that occurred at least 12 months prior to the study and was diagnosed by a physician or a physical therapist based on clinical examination28, (ii) history of at least two episodes of ‘giving way’ (regular occurrence of uncontrolled and unpredictable episodes of excessive inversion of the rear foot) and feelings of ankle joint instability, (iii) the most recent injury occurred more than 6 weeks prior to study enrollment, (iv) answering “yes” to at least five yes/no questions of the Ankle Instability Instrument developed by Docherty et al.29. This should include the first question: “Have you ever sprained your ankle?” and at least four other questions related to the severity of ankle symptoms, and (v) able to bear full weight on the injured lower extremity with no more than mild discomfort. The control group included healthy participants with no history of ankle sprain. Exclusion criteria for all groups were a history of ankle fracture, other pathological conditions or surgical procedures in the lower extremity and vestibular or neurological disorders. Participants were recruited from a university setting and provided written informed consent prior to participating in the study. Ariel University Institutional Review Board approved the study.The sample size for this study was determined based on a power analysis calculation that was conducted using G*Power version 3.130. To normalize the gait cycle, gait events were identified automatically, as suggested by Zeni et al.31 and De Asha et al.32.The study was conducted during one visit at the Neuromuscular and Human Performance Laboratory, at Ariel University. Gait was evaluated under both FW and BW walking conditions, while participants walked on a treadmill . Participants were given standard instructions to walk at their comfortable, self-selected pace. Before data collection, subjects were provided with an opportunity to habituate to walking on the treadmill. They walked barefoot and wore tight, black sports pants and t-shirts. To capture gait data, markers were placed directly on the skin using double-sided tape. A total of 15 reflective markers were placed on each side of the participant’s iliac crest, anterior superior iliac spine, posterior superior iliac spine, greater trochanter, lateral and medial femoral condyles, tibial tuberosity, ankle medial malleolus, ankle lateral malleolus, heel, first toe metatarsal head, first toe metatarsal base, fifth toe metatarsal head, fifth toe metatarsal base, and second and third metatarsal base. In addition, cluster markers were placed at mid-thigh and mid-calf. A six-camera motion capture system  sampled at 250 Hz was used to obtain three-dimensional ankle kinematics and the spatiotemporal data. Data was exported to Visual 3-D software , and processed through a 6-degree of freedom anthropometric model. Ankle angles during walking were calculated using the cardan rotation sequenceUnder each walking condition, 17 consecutive strides were recorded for each participant. Then, the first and last strides were omitted, and the remaining 15 strides were analyzed. In the CAI group, the tested limb was the involved limb. The limb used for analysis in the control group was matched to the CAI by side (right or left). The spatiotemporal outcomes examined were gait speed (m/sec), cadence (steps/min), the percent of the gait cycle spent in double limb support (%DLS), and stride time variability (100 × [standard deviation of stride time/mean stride time]). Outcomes of ankle kinematics included the average and 95% confidence interval (CI) of sagittal and frontal ankle angle throughout the gait cycle.14. A significant difference was defined in case non-overlapping CI was found. In addition, a two-way repeated measures ANOVA using Statistical Parametric Mapping (SPM) was used to analyze the effects of group, condition and interaction (group x condition) of the kinematic data. Significance was determined as P < 0.05. The analysis was conducted using IBM SPSS, v24.0  and the SPM1D v.0.4 package for Python 3.733.Descriptive statistics included mean and standard deviations (SD). Normal distribution of continuous data was verified using Shapiro–Wilk test. Simple chi-square and t-tests were used to compare baseline characteristics between the CAI and control groups. A two-way linear mixed model was performed for each spatiotemporal gait outcome with the factors of group  and walking condition . The interaction effect was evaluated to determine if there were differences between groups in their walking adaptation from forward to backward. To analyze the kinematic parameters, mean sagittal and frontal ankle angles were plotted throughout the gait cycle with their corresponding 95% CI, as was previously describedSubject characteristics are summarized in Table The changes in spatiotemporal characteristics from FW to BW in both groups for all four spatiotemporal gait outcomes, and the results of the linear mixed model are presented in Table The analysis showed a significant effect of condition for gait speed, cadence, and stride time variability. During BW, both groups had slower gait speed (p < 0.001), lower cadence (p < 0.001), and higher stride time variability (p < 0.001). However, there was no significant group or interaction effects for these parameters, indicating no difference between groups in their adaptation to BW. In addition, no between-condition difference was evident in %DLS, as well as no between-group difference in both conditions (FW and BW).Figure The current study found that under BW condition, spatiotemporal and ankle kinematics gait characteristics of subjects with CAI and healthy controls were significantly different when compared to FW. For example, during BW, gait speed was reduced, whereas stride time variability and ankle dorsiflexion were increased. Yet, while major differences were found between BW and FW, there were no differences between groups. These indicate that during BW, subjects with CAI adjust their spatiotemporal and ankle kinematics characteristics similar to the way healthy controls do. To the best of our knowledge, this study is the first to document these results.13, our findings are consistent with previous reports that did not find differences between individuals of these two groups. Two recent reviews indicated that postural stability assessments using a stable surface with the eyes open may not always discriminate between individuals with CAI and healthy controls34. De Noronha and colleagues35 reported no differences in proprioception or motor control between CAI and a control group. A systematic review with meta-analysis conducted to determine the ability of functional performance tests to differentiate between individuals with CAI and healthy controls, concluded that clinical implementation of these tests should be limited, due to inconsistent results36.While several studies have clearly demonstrated substantial differences in movement analysis between subjects with and without CAI6. Yet, conflicting results were reported in other studies where CAI subjects were not found to have more inversion37 or even have greater rearfoot eversion38. Similarly, the results of the present study did not observe increased ankle inversion in the CAI group in FW or BW, as compared to controls.Furthermore, one of the most common characteristics that has been reported in the literature that differs between patients with CAI and healthy participants is greater inversion of the foot relative to the tibia during walking39 recently presented an updated model of CAI. According to this model, there is a list of impairments that people with CAI as a group are likely to demonstrate; however, each individual may present certain clinical and performance outcomes that are affected by personal and environmental factors. It seems that the inconsistencies between studies may be partially explained by this model.Several explanations may be suggested for the inconsistencies observed between studies. It is possible that some discrepancies were due to the heterogeneity of the CAI population. Hertel and Corbett 27. However, according to the Ankle Instability Instrument, only 3 of 16 participants reported that they feel unstable while walking on a flat surface. Previous studies have shown that only very complex walking situations, such as walking with a cognitive dual task, may differentiate CAI subjects from controls 16. Thus, it is possible that while BW on a treadmill required some level of adaptation from both groups, it was not challenging enough to discriminate between their gait performance. Assessments with more challenging tasks, such BW with dual task and BW at fast speed may be more appropriate for testing gait impairments related to CAI.Specifically, all the CAI participants in the current study met established standards for CAI 41. Likewise, previous research with CAI participants has shown that gait outcomes vary when data are collected during barefoot walking44 or with shoes46. For example, Herb et al.45 evaluated gait kinematics while the subjects wore shoes and reported on differences in shank-rearfoot coupling between CAI and control groups across gait cycle. The authors explained the results by altered sensorimotor function in the CAI group due to their ankle pathology. In contrast, our results did not demonstrate differences in movement analysis between subjects with and without CAI, even under a task that requires greater sensorimotor activation such as BW. A possible explanation for this difference may be related to the uniqueness of barefoot walking. Barefoot walking promotes higher plantar loading, resulting with enhanced afferent feedback of proprioception, which is desirable for control of gait and kinematic adjustments. Furthermore, BW walking relies more on proprioception rather than on visual feedback. Thus, the augmented feedback provided by the barefoot walking may increase the ability of the sensorimotor system to organize movement patterns.Another aspect that may have affected the results is related to the procedure of data collection. In the present study, data were collected while subjects were barefoot, as this state detects frontal plane kinematics more accurately. Systematic reviews indicated significant differences in kinematics, kinetics and muscle activity during barefoot and shod walking and running47. Consistent with previous research, BW was characterized by slower gait velocity, reduced cadence, increased ankle dorsiflexion and decreased plantar flexion20. The present study also documented increased stride time variability, which may indicate less stability during BW48.Although BW did not distinguish between groups, it affected the spatiotemporal and kinematic variables in both groups, compared to FW. This finding is in agreement with previous studies that reported changes in spatiotemporal and kinematic characteristics in young adults during BW, as compared to FW50. However, current evidence suggests that BW utilizes additional elements, presumably supraspinal, in addition to a common spinal drive22. The significant adaptations during BW in both groups, and particularly the increased stride time variability, may support the notion that control of BW mechanisms may require more central nervous system resources than does FW.Until recently, BW was considered to be a simple reversal of FW. It was hypothesized that a single spinal mechanism controls both FW and BW5. In the current study, the dorsiflexion peak ankle during FW was similar to findings of previous studies with CAI43 with no difference between groups, but greater dorsiflexion (+ 4.31°) was observed under the BW condition. Emerging research suggests that BW can improve locomotion in patients with neurological lesions, as well in patients with musculoskeletal disorders. A recently published study reported the effectiveness of BW as a rehabilitation technique for patients after anterior cruciate ligament reconstruction51. Based on our results, clinicians may consider training subjects with CAI under varied BW conditions in order to enhance their sensorimotor control of ambulation. Furthermore, the increased ankle dorsiflexion during BW may suggest that this condition can be utilized to gain greater ankle dorsiflexion. To the best of our knowledge, there is no published data to document the effectiveness of BW training for patients with CAI. Thus, future research should be performed to confirm the effectiveness of this intervention.Another interesting finding of the current study is related to sagittal ankle kinematics. Subjects with CAI were reported to have decreased peak ankle dorsiflexion during FW walking compared to healthy controls52. In contrast, during treadmill walking, the speed is fixed. The constant speed and rhythm during treadmill walking may influence the spatiotemporal and kinematics variables. However, while changes in spatiotemporal variables and hip and knee kinematics were demonstrated, ankle kinematics seem to be similar under forward self-selected walking over ground and on a treadmill55. This may support the ecological validity of the findings regarding ankle kinematics during FW in the current study.In the present study, gait was evaluated while the subjects walked on a treadmill. When walking over-ground, a constant speed is not usually sustained for a long period of timeAs far as we know there are no studies that compared BW over a treadmill to BW over ground. Visual information is important for maintaining equilibrium and stability during locomotion. While walking backward visual information is limited and the subject cannot observe potential obstacles. Furthermore, during over-ground locomotion the subject moves with respect to the surroundings, while during treadmill walking the opposite occurs, and the surroundings moves with respect to the subject. This may add more complexity to visual perception during BW treadmill walking. Thus, future studies that will compare BW over ground and on a treadmill are warranted.39. Thus, further investigations with a larger cohort should be undertaken to confirm the study results and assess and analyze relevant subgroups of patients with CAI.This study had several limitations. It was originally powered to identify differences between the groups for gait speed and did not account for the additional spatiotemporal and kinematic variables. Additional limitations are that we did not separate CAI subjects according to mechanical and functional instabilities, and the subjective report of perceived instability during BW was not tested. Updated models of CAI indicate differences between mechanical and functional instability among individuals with CAI and stresses the importance of evaluating self-reported perceived instabilityParticipants with CAI and healthy controls demonstrated significant changes in spatiotemporal and ankle kinematics gait characteristics between BW and FW conditions. However, there were no significant between-group differences in both conditions, indicating that subjects with CAI adjust their spatiotemporal and ankle kinematics characteristics during BW similar to the way healthy controls do. Clinicians should consider this information with caution when assessing and designing training programs for individuals with CAI, due to the heterogeneity of this population.


The plant hormone auxin acts as a signaling molecule to regulate a vast number of developmental responses throughout all stages of plant growth. Tight control and coordination of auxin signaling is required for the generation of specific auxin‐response outputs. The nuclear auxin signaling pathway controls auxin‐responsive gene transcription through the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F‐BOX pathway. Recent work has uncovered important details into how regulation of auxin signaling components can generate unique and specific responses to determine auxin outputs. In this review, we discuss what is known about the core auxin signaling components and explore mechanisms important for regulating auxin response specificity. A review of recent updates to our understanding of auxin signaling. Arabidopsis contains 22 full‐length The complexity of diverse auxin signaling responses can, at least in part, be regulated by unique characteristics imparted by three distinct protein domains within ARF family members—the N‐terminal DNA‐binding domain (DBD), the variable middle region (MR) associated with activating or repressing activity, and the C‐terminal PB1 domain homologous to those found in Aux/IAA repressor proteins  within auxin response genes.Sequence‐specific recognition and binding of the ARF transcription factors to auxin response genes is carried out by the DBD. Structural studies of the ARF1 and ARF5 DBD have revealed a composition of three distinct substructural components.AuxREAuxRE.AuxRE variants must still be validated, however, the presence of multiple target sites with varying ARF binding affinities may begin to help explain differences in ARF activity.Several ARF proteins were initially identified based on their ability to bind the canonical TGTCTC An additional layer of auxin‐responsive gene regulation by ARF transcription factors comes from the ability of these proteins to dimerize through the DD. The DBD of ARF1 and ARF5 form homodimers within the crystal structure with the B3 domains binding to an inverted repeat of the canonical TGTCTC AuxRE element.AuxRE site leads to reduced binding affinity, suggesting that dimerization of ARF proteins leads to cooperative binding at target sites.AuxREs with different spacing.Dimerization of ARF proteins allows for multi‐site recognition of auxin responsive motifs within target genes. Mutation of a single 4.2Whereas structural studies of the ARF DBD and PB1 domains have guided our understanding of the function of these domains, much less is known about the properties of the ARF middle region. This region has the most highly diverged amino acid composition and length amongst ARFs and thus it has been difficult to tease apart contributions of the MR in regulating ARF activity. The ARF MR alone is sufficient to confer transcriptional activator or transcriptional repressor activity.in planta.Although the precise mechanisms that regulate ARF activator and repressor activities are unclear, the MR of ARF proteins likely contain an intrinsically disordered region (IDR) that confers these activities  that allow for auxin responsiveness.M. polymorpha to those of plant lineages with expanded auxin signaling protein families, such as P. patens and C. richardii, revealed that the number of ARF transcription factors scales with the number of auxin‐regulated genes.The presence of all necessary auxin signaling components can be found in the common ancestor of land plants, however, another important question is how this signaling pathway evolved in complexity to enable diverse auxin responses. Arabidopsis accessions have revealed interesting insights. These studies have uncovered evolved sequence variations that correspond to altered molecular phenotypes and underscore how small changes can have significant impacts on protein function and consequent auxin response.Not only have auxin signaling components been examined across evolutionary history,6Cis‐trans isomerization of prolines in Aux/IAA proteins is involved in auxin response, perhaps by regulating recognition by the SCF complex.Post‐translational modifications of each of the core auxin signaling components impart an additional level of regulation to auxin response outputs Table . For exaAlthough many of these modifications and their roles in regulating auxin outputs need to be confirmed in vivo, ARF7 and ARF19 are phosphorylated by the BRASSINOID‐INSENSITIVE 2 (BIN2) kinase to regulate lateral root organogenesis.BIN2‐mediated phosphorylation of ARF7 and ARF19 results in relief from Aux/IAA repression and enhances transcriptional activity by these ARF transcription factors.In addition, SUMOylation of ARF7 plays a role in hydropatterning of lateral roots.7Within the past several years, an increasing number of studies have begun to highlight the importance of biomolecular condensates as a means to regulate diverse biological functions within the cell . ARF3 does not contain a PB1 domain (reviewed in Refs. 9Taken at face value, the auxin signaling pathway seems a fairly straightforward mechanism for auxin perception and response; however, we are only beginning to understand the multiple layers of regulation necessary for generating distinct and dynamic auxin outputs. Diversity in signaling component family members allows for different combinations of protein interactions. Additionally, post‐translational modifications, establishment of transcriptional regulatory complexes, and interactions with components from other signaling pathways may play a role in auxin response specificity. Transcriptional regulation of auxin response components,The authors declare no potential conflict of interest.


Type III CRISPR-Cas systems, which are widespread in both bacteria and archaea, provide immunity against DNA viruses and plasmids in a transcription-dependent manner. Since an unprecedented cyclic oligoadenylate (cOA) signaling pathway was discovered in type III systems in 2017, the cOA signaling has been extensively studied in recent 3 years, which has expanded our understanding of type III systems immune defense and also its counteraction by viruses. In this review, we summarized recent advances in cOA synthesis, cOA-activated effector protein, cOA signaling-mediated immunoprotection, and cOA signaling inhibition, and highlighted the crosstalk between cOA signaling and other cyclic oligonucleotide-mediated immunity discovered very recently. CRISPR-Cas systems are known to provide adaptive immunity against viruses and plasmids in prokaryotes. Based on the composition of effector complexes, CRISPR-Cas systems were divided into two classes which could be further subdivided into six types (types I–VI) and multiple subtypes . Class 1The Cas 10 subunit of type III effector complex and the ancillary ribonuclease Csm6/Csx1 are two important components involved in cOA signaling. Cas 10 contains an N-terminal histidine-aspartate (HD) domain and two Palm domains with a GGDD motif inserted into the second Palm domain . The HD Cas10 is the largest subunit of type III effector complex and is a signature protein of type III systems . The cOAStreptococcus thermophilus and Enterococcus italicus, respectively , which forms hydrogen bonds with base of ATP and confers specificity for ATP  protein, AcrIII-1, was recently identified as a ring nuclease that specifically degrades cOAimmunity . AcrIII-proteins ,c. AcrIIproteins . Very reimmunity .4. However, given that Cas10 synthesizes cOAs ranging from cOA3 to cOA6, it is rational to predict the existence of other cOA-specific nucleases in prokaryotes and viruses. A cOA6-activated Csm6 can cleave cOA6 by its CARF domain  specifically degrade cOAF domain , indicatous cOAs . Additioous cOAs . The inh3-activated DNA endonuclease in CBASS is also present in some type III systems and is considered to be an important effector protein of cOA signaling for immunity against phage (3) which are synthesized by CD-NTases (In recent studies, a large family of CD-NTases have been found to produce a wide variety of cyclic di- and trinucleotides including 3'3' cyclic UMP-AMP, 3'3'3' cyclic AMP-AMP-GMP, and 2'3'3' cyclic AMP-AMP-AMP, which had never been reported previously . These cst phage . Very reD-NTases . InteresD-NTases , which mCyclic oligonucleotide molecules discovered in the past 3 years have greatly expanded our understanding on nucleotide signaling molecules in prokaryotes for decades. Due to the diversity of the effector proteins in cOA signaling and crosstalk between cOA signaling and CBASS, cOA signaling is far from fully elucidated. Future studies of cOA regulation will further expand our understanding of the role of cOA signaling and give us new insights into the cyclic oligonucleotides involved in antiviral defense systems.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Human T-cell leukemia virus type 1- (HTLV-1-) associated arthritis is a relatively common disease. However, tenosynovitis owing to adult T-cell leukemia-lymphoma (ATL) is a rare condition. To the best of our knowledge, there have been no reports of tenosynovitis caused by conversion to acute ATL from one of the other ATL types. We present the case of a 60-year-old woman with rheumatoid arthritis (RA) with bilateral wrist tenosynovitis owing to the conversion to acute ATL from one of the other ATL types. She had swelling around the bilateral wrist joint under well controlled RA inflammation. She had no symptoms, physical findings, or laboratory findings indicative of conversion to acute ATL from one of the other ATL types. She underwent tenosynovectomy on the volar and dorsal sides of the left wrist joint to diagnose the cause of swelling around the bilateral wrist joint. Pathological analysis revealed diffuse proliferation of medium-sized atypical CD4(+) lymphocytes. Interestingly, she was diagnosed with wrist tenosynovitis caused by an acute ATL type. This diagnosis suggested that clinicians must consider ATL in connection with atypical wrist tenosynovitis in HTLV-1-endemic areas. Adult T-cell leukemia-lymphoma (ATL) is a peripheral T-cell malignancy associated with human T-cell leukemia virus type 1 (HTLV-1). ATL is classified into four clinical types: acute, lymphomatous, chronic, and smoldering . A previThe study protocol adhered to the ethical guidelines of the 1975 Declaration of Helsinki, and the study was approved by the institutional review board of our institute. The patient provided informed consent for publication of the case report and the associated images.3 with 5% abnormal lymphocytes, serum lactase dehydrogenase levels of 224 IU/L , serum calcium levels of 9.9 mg/dL , serum albumin levels of 4.0 mg/dL , and no tumor lesion [The patient was a 60-year-old woman who presented to the Rheumatology Department of our hospital in November 2013 with polyarthralgia  joint of the right middle and ring fingers, bilateral knees, and bilateral foot joints) and morning stiffness in both hands. She was diagnosed with seropositive RA (rheumatoid factor- (RF-) positive, 44 IU/mL; anticyclic citrullinated peptide antibody-negative, <0.6 IU/mL) according to the 2010 American College of Rheumatology/European League Against Rheumatism . Thereaf3 with 37.5% abnormal lymphocytes; serum lactase dehydrogenase levels, 269 IU/L; serum calcium levels, 8.9 mg/dL; serum albumin levels, 3.8 mg/dL; C-reactive protein (CRP) level, 0.02 mg/dL ; erythrocyte sedimentation rate, 6 mm/h ; serum matrix metalloprotease-3 (MMP-3) levels, 57.4 ng/mL ; and soluble interleukin-2 receptor levels, 2999 IU/mL . Anterior-posterior plain radiographs of the hands showed bone atrophy around the wrist and MP joint , and neurolysis of the median nerve. Surgical examination of the volar side revealed massive tenosynovitis around the flexors and compression and adhesion of the median nerve owing to tenosynovitis . SurgicaPathological analysis revealed the diffuse proliferation of medium-sized atypical lymphocytes. The surface marker profile of the atypical lymphocytes in the tenosynovial tissue was as follows: CD3(+), CD4(+), CD8(-), CD20(-), CD25(+), CD30(-), CC chemokine receptor 4(+), and Foxp3(+). The lymphohistiocytic infiltration was positive for Ki-67 . The patDeltaretrovirus, family Retroviridae, and subfamily Orthoretrovirinae [α treatment combined with zidovudine or watchful waiting is recommended for symptomatic chronic-type and smoldering-type ATL, and watchful waiting alone is recommended for asymptomatic chronic-type and smoldering-type ATL [α treatment combined with zidovudine was highly effective and significantly improved the survival rates in patients with chronic and smoldering ATL types [α combined with zidovudine before acute conversion of the smoldering ATL type.The number of HTLV-1 carriers is approximately 10 million people worldwide, with endemic foci in the Caribbean Basin, Latin America, intertropical Africa, Melanesia, and Japan [ovirinae . HTLV-1 ovirinae , 12, 13.ovirinae . Based oovirinae . As the ovirinae . Interfetype ATL . Howevertype ATL . In addiTL types . Therefoα), which causes synovial proliferation [Past studies have reported a relationship between HTLV-1 and arthritis. In particular, HTLV-1 has been found in the synovial fluid and tissue ; HTLV-1-feration . Moreoveferation , 22. Theferation –6, 21. Hferation , 23. A pferation . Many stferation –6, 19–23feration , 8. Our The patient's independent risk factors for developing ATL were the high HTLV-1 proviral load, older age, family history of ATL, and initial HTLV-1 infection during treatment for other diseases . InteresIn conclusion, ATL is a rare cause of wrist tenosynovitis; however, clinicians must consider it in connection with the atypical wrist tenosynovitis in HTLV-1-endemic areas.


It has become increasingly apparent that G protein-coupled receptor (GPCR) localization is a master regulator of cell signaling. However, the molecular mechanisms involved in this process are not well understood. To date, observations of intracellular GPCR activation can be organized into two categories: a dependence on OCT3 cationic channel-permeable ligands or the necessity of endocytic trafficking. Using CXC chemokine receptor 4 (CXCR4) as a model, we identified a third mechanism of intracellular GPCR signaling. We show that independent of membrane permeable ligands and endocytosis, upon stimulation, plasma membrane and internal pools of CXCR4 are post-translationally modified and collectively regulate EGR1 transcription. We found that β-arrestin-1 (arrestin 2) is necessary to mediate communication between plasma membrane and internal pools of CXCR4. Notably, these observations may explain that while CXCR4 overexpression is highly correlated with cancer metastasis and mortality, plasma membrane localization is not. Together these data support a model where a small initial pool of plasma membrane-localized GPCRs are capable of activating internal receptor-dependent signaling events. DeNies et al. identify a new mechanism of intracellular GPCR signalling. Using CXC chemokine receptor 4 (CXCR4) as a model, they show that upon stimulation with receptor agonists that not only plasma membrane-localized receptors, but also intracellular CXCR4 molecules are post-translationally modified and regulate transcription. This study suggests that a small pool of plasma membrane-localized GPCRs can activate internal receptor-dependent signaling, and that β-arrestin-1 mediates this activation. Extensive research has illustrated that GPCRs elicit distinct signaling events at different plasma membrane micro-domains, as well as endocytic compartments that are important for cell physiology and disease pathogenesis9. These studies support a model where the location, in addition to magnitude, of a signaling event is important for cellular decision-making. Others have shown that GPCR site-specific posttranslational modifications (PTMs) modulate adaptor protein recruitment, GPCR localization, and consequently receptor-signaling events12. Together these observations motivated us to reexamine some confounding observations pertaining to the relationship of receptor localization, PTM, and signaling for CXC chemokine receptor 4 (CXCR4).While extracellular inputs, cell membrane receptors, and resulting transcriptional programs are diverse, many receptor-signaling events converge to a reduced number of signaling hubs. Cellular mechanisms that mediate this process, as well as strategies to control these actions remain outstanding questions. Over the last decade, we have learned that G protein-coupled receptor (GPCR) spatiotemporal signaling is one mechanism used by cells to translate diverse environmental information into actionable intracellular decisions while using seemingly redundant signaling cascades16. It is deregulated in 23 different cancers and overexpression is often correlated with metastasis and mortality21. However, surprisingly, plasma membrane expression is not correlated with metastasis21 and in some cancer tumor specimens, as well as cell culture models, samples with poor CXCR4 plasma membrane localization remain responsive to CXCR4 agonist26. CXCR4 is activated by a highly receptor-specific 8 kDa chemokine, CXCL12 (refs. 29). Unlike β-adrenergic receptors, which have been shown to be activated at intracellular compartments in an OCT3 cationic transporter-dependent mechanism7, endocytic-independent internalization of CXCL12 is unlikely due to its size. Given that receptor activation is dependent on ligand binding or transactivation by another receptor31, the aforementioned observations are confounding, as cells with low plasma membrane CXCR4 remain highly responsive to CXCL12. There are two potential explanations for this observation. Firstly, this could be due to spare receptors on the plasma membrane as it is well established that only a limited number of plasma membrane receptor contribute to signaling32. Alternatively, this could be due to activation of intracellular pools of receptors.CXCR4 is a type 1 GPCR that regulates a variety of biological processes, such as cell migration, embryogenesis, and immune cell homeostasis33. This commercially available antibody is raised against the C-terminus of the receptor, and upon CXCL12 stimulus quickly loses its ability to detect CXCR4 due to receptor PTM33. To attempt to identify the specific PTMs responsible, we treated lysates from WT and ubiquitination mutant receptor-expressing cells with phosphatase. While phosphatase treatment ablated AKT S473 phosphorylation, no change in UMB2 detection was observed  as a robust tool to study CXCR4 PTM33. We found that by mutating C-terminal lysine residues to arginine (K3R), CXCR4 plasma membrane localization was reduced by >50%  restored receptor plasma membrane localization to near WT levels  cells Fig. . We chos50% Fig. . While tels Fig. . Interesels Fig. . In accoels Fig. 38,39. Welgi Fig. . Non-plaell Fig. . To explell Fig. . This su37. Since receptor PTM plays a role in mediating receptor signaling, we investigated the effect of receptor localization on agonist-induced CXCR4 PTM. Although UMB2 detection continued to decrease over a course of 3 h  pools of CXCR4 could be posttranslationally modified in response to agonist stimulation and contribute to signaling. To investigate this, we examined the localization of CXCR4 PTM during receptor signaling. As previously observedied Fig. . To testied Fig. . Receptoied Fig. . To ensuied Fig. . To furtied Fig. . While tied Fig. , agonistied Fig. . Togethe45. Upon ligand binding, G protein Gαi and Gβγ subunits are released from the GPCR due to guanidine exchange factor activity. Gβγ activates GPCR kinases (GRKs), which quickly phosphorylate the C-terminus of activated receptors leading to β-arrestin recruitment  that have preexisting signaling defects  transcript levels upon CXCL12 stimulus in RPE cells with high and low plasma membrane CXCR4 expression. EGR1 transcription is downstream of the ERK1/2 pathway and has been shown to be induced by CXCL12 (refs. 51). As expected, WT RPE cells (not overexpressing CXCR4) were unresponsive to CXCL12  of CXCR4 are being posttranslationally modified. While agonist-induced internal CXCR4 PTM appear to partially occur at the Golgi Fig. , CXCR4 m56. AMD3100 (Plerixafor) is the only CXCR4 inhibitor that, to our knowledge, is FDA approved57. This inhibitor is membrane permeable and ubiquitously inhibits CXCL12-induced signaling by preventing interactions between CXCL12 and CXCR4 (ref. 57). Since CXCR4 plays an important role in multiple cellular processes, the ability to selectively target different populations of CXCR4 might lead to more successful therapies that do not act as a blunt instrument, which ubiquitously disrupt downstream signaling activity from the inhibited receptor. Similarly, significant recent research has led to the development of biased peptide agonists and antagonists for CXCR4, as well as other GPCRs56. The goal of these therapies is to selectively modulate receptor-signaling pleiotropism56. To our knowledge, these efforts have largely focused on modulating signaling of plasma membrane receptors. However, coupled with our findings, it would be interesting to explore whether intracellular targeting can improve their therapeutic utility. Additional research exploring localized receptor signaling is necessary to truly elucidate the relationship between localization and signaling. We believe this new paradigm has the potential to influence the design of next-generation therapies.It is now widely accepted that GPCRs signal from multiple intracellular localizations and that the localization of GPCR activation differentially impacts downstream signaling. Consequently, it is plausible that aberrant signaling from different cellular compartments is associated with disease. Thus, inhibiting receptor signaling at different cellular compartments may be an effective therapeutic strategy that reduces side effects. While there has been great interest over the last few decades to modulate CXCR4 function in both HIV and cancer therapeutics research, therapeutic success has been limitedβγ and β-arrestins in regulating GPCRs, and support a new model of GPCR signaling whereby plasma membrane and internal pools of receptors communicate to collectively determine a cellular response. These observations may resolve the paradox that while CXCR4 overexpression is associated with metastatic potential, plasma membrane localization is not. In addition, this work supports a growing amount of evidence supporting that targeting specifically intracellular GPCR populations or the downstream signaling cascades activated by these pools might lead to improved therapeutic strategies for treating cancer, cardiovascular disease, and pain management40.While many questions remain, the data presented expand the role of GLiCor Odessey CLX & SA Imagers, Azure Sapphire 4 laser Imager, and BioRad RT-qPCR ThermoCycler.HeLa cells were originally obtained from ATCC. HeLa cells were cultured in DMEM media (Corning) supplemented with 10% FBS (Corning). RPE cells were a gift from Dr. Sandra Schmid at UT Southwestern. All stable cell lines were directly derived from this RPE line. RPE cell lines were cultured in DMEM/F12 media (Corning) supplemented with HEPES, glutamate, and 10% FBS (Corning). HEK293T cells were obtained from ATCC and grown in DMEM (Corning) media supplemented with 10% FBS.33. K3R and K3R/Q mutant receptors were generated by PCR mutagenesis of WT CXCR4 in the pLVX plasmid, using the NEB Quick-change mutagenesis kit. The low plasma membrane CXCR4 construct was generated by PCR amplification  and restriction enzyme cloning, using the BsrRI and EcoRI restriction enzymes. All CXCR4 constructs had an N-terminal FLAG tag and C-terminal MYC tag for easy antibody detection. Stable cell lines expressing WT and mutant CXCR4 receptors were generated by lentiviral transduction. Lentiviruses (shRNA and CXCR4 constructs for stable cell lines) were generated by co-transfecting HEK293T cells with the pLVX transfer plasmid, psPAX2, and pMD2.G lentiviral envelope and packaging plasmids. To generate stable cell lines, supernatant media containing mature lentiviral particles was collected 4 days post transfection and added to RPE cells, and cells stably expressing the constructs were generated via puromycin selection (3 µg/ml). All transfections were conducted using Lipofectamine 2000 (Life technologies).WT CXCR4 was generated as previously described33. For intracellular staining, cells were first disassociated using 50 µM EDTA in Ca2+-free PBS and fix for 10 min in 4% paraformaldehyde at room temperature. Afterward, cells were permeabilized using 0.2% Triton-X 100 for 10 min at room temperature. Intracellular targets were labeled with primary antibodies for 1 h at room temperature after which cells were washed with PBS and incubated with secondary antibodies for 1 h at room temperature—see Table 33. Representative flow cytometry data collected can be found in Supplementary Fig. Flow cytometry experiments for plasma membrane receptor labeling were conducted, as previously describedg for 45 min at 4 °C. Afterward, lysates were immediately stored at −20 °C or processed for immunoprecipitation or western blotting.Cells were seeded in 12-well plates 24 h prior to each signaling experiment achieving 70–80% confluence at the experimentation time. Cells were serum-starved in DMEM/F12 media without FBS for 4 h prior to each signaling experiment. For inhibitor experiments , cells were pretreated for 30 min with the respective inhibitors and throughout the signaling experiment. For shRNA experiments, cells were transduced with either scramble or β-arrestin-1 or 2 shRNA (Table g) and internal (non-plasma membrane) proteins collected by removing the supernatant. To prevent potential biotinylated protein contamination, only 200 µl of supernatant was removed. Afterward, beads were washed three times with RIPA buffer containing protease inhibitors. After the final wash, all buffer was removed.For plasma membrane biotinylation experiments, biotinylated plasma membrane proteins were isolated from WCL using high capacity streptavidin agarose beads Table . Approxi33. Specific normalization procedures for each experiment are described in the respective figure legends. All statistics were calculated using two-tailed t tests. Complete western blots shown in Supplementary Figs. Prior to western blotting, samples were incubated with Laminelli buffer supplemented with β-mercaptoethanol (loading buffer). For surface biotinylation samples, β-mercaptoethanol concentration was increased twofold and samples were incubated at room temperature in the loading buffer for 30 min prior to western blotting to denature proteins from beads. Samples were run on SDS–PAGE 4–20% BioRad gels (15 well/15 µl or 10 well/50 µl gels). For all signaling experiments, 12.5 µl of lysate was loaded while for surface biotinylation assays, 35 µl of lysate was loaded. SDS–PAGE gels were run at constant 140 V for ~60 min. Afterward, proteins were transferred to PVDF membranes using the iBlot transfer systems (mixed range proteins 7 min setting) and membrane incubated in blocking solution (1% BSA in TBST) rocking for 1 h at room temperature. Afterward, blots were incubated with their respective antibodies  for the respective time courses shown in the figure legends. RNA was extracted using the Zymogen RNA extraction kit (R1054) and 1 µM cDNA was synthesized using the iScript synthesis kit (BioRad). qPCR assays were conducted using SYBR Green (BioRad) per BioRad protocol instructions, using 12.5 ng of cDNA for each well. Samples were run in duplicate and primers used in this study are shown in Table Cells were seeded in six-well plates on glass coverslips 24 h prior to each experiment and serum-starved for 4 h as described above. Cells were stimulated as specified in each figure legend and immediately washed with PBS and fixed in 4% paraformaldehyde for 10 min at room temperature. Cells were permeabilized for 10 min with 0.2% Triton-X 100 diluted in PBS and subsequently blocked with 2.5% BSA diluted in PBS (blocking solution) for 1 h. Cells were incubated with primary antibody diluted in blocking solution and incubated overnight at 4 °C (Table t test and statistical significance denotes p < 0.05.Throughout the manuscript all individual data points are plotted. Sample mean, median, and standard deviation are also shown on each bar graph. Unless noted in the figure legend, all statistical analyses were conducted using a two-tailed Further information on research design is available in the Peer Review FileSupplemental InformationSupplementary Data 1Reporting Summary


Japan has been running a nationwide antenatal human T-cell leukemia virus type-1 (HTLV-1) antibody screening program since 2010 for the prevention of HTLV-1 mother-to-child transmission. As part of the program, pregnant women are invited to take an HTLV-1 antibody screening test, usually within the first 30 weeks of gestation, during regular pregnancy checkups. Pregnant women tested positive on the antibody screening test undergo a confirmatory test, either western blotting or line immunoassay. In indeterminate case, polymerase chain reaction (PCR) is used as a final test to diagnose infection. Pregnant women tested positive on a confirmatory or PCR test are identified as HTLV-1 carriers. As breastfeeding is a predominant route of postnatal HTLV-1 mother-to-child transmission, exclusive formula feeding is widely used as a postnatal preventive measure. Although there is insufficient evidence that short-term breastfeeding during ≤3 months does not increase the risk of mother-to-child transmission compared to exclusive formula feeding, this feeding method is considered if the mother is eager to breastfeed her child. However, it is important that mothers and family members fully understand that there is an increase in the risk of mother-to-child transmission when breastfeeding would be prolonged. As there are only a few clinical studies on the protective effect of frozen-thawed breastmilk feeding on mother-to-child transmission of HTLV-1, there is little evidence to recommend this feeding method. Further study on the protective effects of these feeding methods are needed. It is assumed that the risk of anxiety or depression may increase in the mothers who selected exclusive formula feeding or short-term breastfeeding. Thus, an adequate support and counseling for these mothers should be provided. In addition to raising public awareness of HTLV-1 infection, epidemiological data from the nationwide program needs to be collected and analyzed. In most cases, infected children are asymptomatic, and it is necessary to clarify how these children should be followed medically. While the majority of HTLV-1-infected individuals remain asymptomatic, the two well-recognized disease associations ATL and HAM/TSP are caused by the virus. HTLV-1 carriers are estimated to have a lifetime risk of 2–7% for the development of ATL  and 0.25A first step in taking measures to prevent HTLV-1 MTCT is to determine whether the mother is infected. To date, there are no effective measures to prevent antenatal infection, but avoiding or restricting breastfeeding is expected to reduce the number of postnatal infections via MTCT. In turn, the prevalence of HTLV-1-associated diseases could be reduced, and the rising trend in the number of people with horizontal infection could be curbed to some extent. Non-endemic and endemic countries may have different views on the need to introduce a nationwide screening program, but in countries or areas where HTLV-1 is endemic, antenatal screening is likely to contribute to a reduction in the burden of associated diseases .In 2010, the Ministry of Health, Labor, and Welfare in Japan decided to conduct a nationwide HTLV-1 antibody screening program for all pregnant women . Japan iThe United Kingdom National Screening Committee had considered antenatal HTLV-1 screening program three times, but the committee did not recommend introducing a screening program in the United Kingdom because of the low prevalence of HTLV-1 infection and the low risk for infected infants to develop a serious illness. The Committee maintained its conclusions after updating and reviewing the evidence in 2017 . HoweverIn this review, we would like to introduce the nationwide antenatal screening program in Japan and discuss the associated issues.The algorithm for HTLV-1 carrier screening during pregnancy in Japan is shown in In Japan, laboratory screening for HTLV-1 infection has been routine practice for blood donors since 1986 . FurtherSeveral assays for HTLV-1 antibody screening are available, including PA , CLEIA , CLIA Q, and ECLHowever, antibody screening tests use different antigens and have different measurement principles, and the test results often do not match between them due to the methods used. In addition, these tests have a high false-positive rate, especially in non-endemic areas. For this reason, a confirmatory test must be performed following a positive screening test.According to data collected retrospectively by the Japan Association of Obstetricians and Gynecologists, the prevalence rate of pregnant women tested positive on a PA or CLEIA screening test was 0.32%  in 2011. Among 2,259 pregnant women who screened positive, 1,894 women (83.8%) underwent a WB test as a confirmatory test. Thus, the screening program was still in its early days, and confirmatory tests were not performed on all cases.The number of WB positive, indeterminate, negative, and missing cases was 942 (49.7%), 212 (11.1%), 660 (34.8%), and 80 (4.2%), respectively. The rate of false-positive results was 14.0% (88/629) in Kyushu and Okinawa prefecture, which are endemic areas in Japan, whereas it was 45.2%  in other areas . The resWestern Bolt is the approach that has been the most frequently used for the confirmatory test. WB measures the serological reaction to both Gag core proteins  and the Env protein gp46 . UnfortuRecently, the LIA has been implemented in Japan in replacement of WB. LIA was developed for the serological confirmation and discrimination of HTLV-1 and -2 infection . This as5PBMCs).However, PCR should be conducted for determining PVL in those cases where the confirmatory tests show indeterminate results. Nowadays, both LIA and qualitative PCR test are covered by the Universal Health Insurance system in Japan as part of the antenatal HTLV-1 screening program. If the PCR qualitative test is negative, it means that there is no infection or that the PVL is below the sensitivity of measurement , but significantly lower than that on longer term (≥6 months) breastfeeding  (The ATL Prevention Program in Nagasaki from 1987 to 2004 showed an 7.4% (15/202) incidence of MTCT in children that were breastfed for <6 months. This was significantly higher than the rate of MTCT on ExFF  . Therefo< 0.001) .P = 0.012; Several studies have shown that the rates of MTCT with ≤3 months of STBF ranged from 0 to 8.5% , while rAs children with longer duration of breastfeeding have higher rates of MTCT , it shouin vitro due to the process of freezing and thawing. The rate of MTCT on FTBMF in previous studies ranged from 0 to 7.1%  Identification of children as carriers will allow minimization of transmission to sexual partners in the future; and (2) If you know that your child is a carrier, you will have immediate access to information when effective treatment strategies for ATL and HAM/TSP become available in the future.While a few patients have severe symptoms, most infected individuals remain asymptomatic throughout their lives and their infections may be unknown to many health providers. In addition, healthcare providers except for specialists have little experience with HTLV-1-associated diseases, and residents have little knowledge about the virus in non-endemic areas. However, as mentioned in an open letter to WHO, “HTLV-1 remains a strong threat to individual and community health, and even more so to global health because of the accelerated rate of human migration in recent times” . AlthougAccording to an interview with thirteen infected people conducted by Little is known about the impact of the diagnosis on the mother’s emotional state (anxiety and depression), their delivery experience or the mother–infant bonding, and the relationship between the mother and her family . Recent In developed countries, it may be possible to adopt ExFF safely for MTCT prevention because the sanitation environment is up to date. On the other hand, infants and children who have received longer term breastfeeding have lower infectious morbidity and mortality, fewer dental malocclusions, and higher intelligence than those who have been breastfed for a shorter period, or not at all. This inequality persists until later in life. Growing evidence also suggests that breastfeeding might protect against a tendency to be overweight and to develop diabetes later in life . A meta-4 PBMC) and ≥ 2 previous HTLV-1-infected children were independently associated with MTCT in a multiple logistic regression. Several risk factors for HTLV-1 MTCT other than long-term breastfeeding are known, including high mother HTLV-1 antibody titers and PVL . Paiva eIn the future, should it become possible to use risk factors to clearly predict the risk of MTCT, it may be possible to reduce the number of children recommended to have breastfeeding avoided or limited.Since the 1990s, ExFF has been used as the main method to prevent postnatal MTCT. Considering the psychosocial influences carrier mothers are subjected to and the potential health risks in their infants and children associated with either completely avoiding or restricting breastfeeding, the development of additional preventive MTCT strategies such as vaccine or antiviral regimens should be developed in the future.In animal experiments, it was reported that the administration of HTLV-1 antibody  and the in utero from carrier pregnant women may offer insufficient protection in very preterm and/or very-low-birth-weight infants. We assume that FTBMF may be safer than feeding with the mother’s own milk without any treatment. However, there are few studies on MTCT in these infants to support this hypothesis.The potential for viral transmission from mother to child presents a dilemma on how best to interpret the benefits and risks of breastfeeding in different settings . Meta-anA pregnant woman with a negative result may become infected from sexual contact with a HTLV-1-infected partner after the screening test, in which case the child could become infected by long-term breastfeeding . If you Not all pregnant women may have been screened for HTLV-1 antibodies during pregnancy, in which case serological antibody testing for such a woman should be performed after delivery. It is unclear whether breastfeeding during a very short period of time before the mother’s test results are obtained will increase the risk of MTCT after birth. In the case there is an infected sibling due to MTCT, the use of infant formula may be an option to minimize the postnatal MTCT risk to the newborn infant until the test results are obtained. Later, if the mother proves to be a carrier, the healthcare provider should discuss feeding methods with her.Adult T-cell leukemia is generally known to be occurred in individuals with vertical infection via mainly prolonged breastfeeding, and HAM/TSP to be occurred in individuals infected via sexual intercourse or blood transfusion during adulthood. Owing to the long latency of the virus, mean onset age in ATL is 66.0 years old . The aveHowever, several studies suggested that children infected via MTCT present with higher risk of developing ATL and/or HAM/TSP in Latin America . KendallIt remains unclear whether the association of skin lesions with HAM/TSP in HTLV-1 infected children is unique to Latin America due to a lack of studies in Japan. Yoshida et al. reported that disease onset was before 15 years of age in 10% of HAM/TSP patients in Japan . These pAs most infected children are asymptomatic, clinic consultation intervals and points of attention at the time of the consultation are unclear. In addition, considering the psychological effects on children, there is some debate about how old it is to be notified them to be infected. Thus, discussions are needed on how to follow up the infected children.In Japan, an antenatal HTLV-1 antibody screening program has been implemented on a nationwide scale for preventing MTCT of the virus. Pregnant women tested positive on a confirmatory or PCR test are identified as HTLV-1 carriers. Since the main infection route of HTLV-1 MTCT is breastfeeding, it is reasonable to recommend avoiding breastfeeding. Nowadays, ExFF has been considered as the most reliable method for MTCT prevention. The STBF during ≤3 months is considered if the mother is eager to breastfeed her child. However, it is important that mothers and family members fully understand not only an increase in MTCT risk with increased duration of breastfeeding but also having an insufficient evidence. As there are only a few clinical studies on the protective effect of frozen-thawed breastmilk feeding on MTCT of HTLV-1, there is little evidence to recommend this feeding method. Further study on the protective effects of STBF and FTBMF are needed.It is assumed that the risk of anxiety or depression may increase in the mothers who selected ExFF or STBF not only during pregnancy but also postpartum. Thus, not only to provide an adequate support and counseling for these mothers in various fields but also to raise public awareness of the risks and prevention methods of HTLV-1 infection is urgently necessary. As most infected children are asymptomatic, further study is needed on how to follow up them.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.All authors contributed to the conception and design of the study, contributed to manuscript revisions, read and approved the submitted version. KI wrote the first draft of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Wnt signaling regulates numerous cellular processes during embryonic development and adult tissue homeostasis. Underscoring this physiological importance, deregulation of the Wnt signaling pathway is associated with many disease states, including cancer. Here, we review pivotal regulatory events in the Wnt signaling pathway that drive cancer growth. We then discuss the roles of the established negative Wnt regulator, casein kinase 1α (CK1α), in Wnt signaling. Although the study of CK1α has been ongoing for several decades, the bulk of such research has focused on how it phosphorylates and regulates its various substrates. We focus here on what is known about the mechanisms controlling CK1α, including its putative regulatory proteins and alternative splicing variants. Finally, we describe the discovery and validation of a family of pharmacological CK1α activators capable of inhibiting Wnt pathway activity. One of the important advantages of CK1α activators, relative to other classes of Wnt inhibitors, is their reduced on-target toxicity, overcoming one of the major impediments to developing a clinically relevant Wnt inhibitor. Therefore, we also discuss mechanisms that regulate CK1α steady-state homeostasis, which may contribute to the deregulation of Wnt pathway activity in cancer and underlie the enhanced therapeutic index of CK1α activators. AxinIn response to Wnt ligands, components of the destruction complex are recruited to the plasma membrane, where axin interacts with LRP5/6 to promote signalosome formation and subsequent activation ,42,43. IIn the absence of nuclear β-catenin, the groucho/transducin-like enhancer of split proteins (Gro/TLE) bind to the T cell factor (TCF)/lymphoid enhancer-binding factor 1 (LEF1) family of transcription factors to repress the Wnt transcriptional program  51]. Up. Up51]. Int1 (Wnt1) was shown to induce mammary adenocarcinoma in mice . H. H123]. Xenopus laevis embryo extracts [maxV, without changing its mK for its substrate [Based on its important negative role in Wnt signaling, pharmacological activation of CK1α should attenuate Wnt activity. The FDA-approved anthelmintic drug pyrvinium has been the first-in-class small-molecule CK1α activator A, havingextracts . Importaextracts ,119. Pyrubstrate ,119. Intubstrate , this reConsistent with the pivotal role of CK1α in Wnt signaling, pyrvinium attenuates the growth of Wnt-dependent CRC cell lines in a CK1α-dependent manner ,96. PyrvA number of potent, chemically novel CK1α activators (SSTC3 and SSTC104- see Although remarkable effort has been made in the identification and development of Wnt pathway inhibitors, the on-target toxicity of these drugs, including the disruption of bone and intestine tissues, has limited their clinical utility. However, the use of additional drugs that ameliorate these on-target toxicities has extended the potential of currently available Wnt-inhibiting drugs ,131,132.As a ubiquitous protein kinase, CK1α plays a role in many other cellular processes in addition to Wnt signaling . For exaIn addition to Wnt- and Shh-driven cancers, CK1α activators have also shown the ability to treat other diseases, such as RAS-driven cancer and Wnt-driven ischemic injury ,136. Thu


The Janus kinase-signal transducer and activator of transcription protein (JAK-STAT) pathway mediates essential biological functions from immune responses to haematopoiesis. Deregulated JAK-STAT signaling causes myeloproliferative neoplasms, leukaemia, and lymphomas, as well as autoimmune diseases. Thereby JAKs have gained significant relevance as therapeutic targets. However, there is still a clinical need for better JAK inhibitors and novel strategies targeting regions outside the conserved kinase domain have gained interest. In-depth knowledge about the molecular details of JAK activation is required. For example, whether the function and regulation between receptors is conserved remains an open question. We used JAK-deficient cell-lines and structure-based mutagenesis to study the function of JAK1 and its pseudokinase domain (JH2) in cytokine signaling pathways that employ JAK1 with different JAK heterodimerization partner. In interleukin-2 (IL-2)-induced STAT5 activation JAK1 was dominant over JAK3 but in interferon-γ (IFNγ) and interferon-α (IFNα) signaling both JAK1 and heteromeric partner JAK2 or TYK2 were both indispensable for STAT1 activation. Moreover, IL-2 signaling was strictly dependent on both JAK1 JH1 and JH2 but in IFNγ signaling JAK1 JH2 rather than kinase activity was required for STAT1 activation. To investigate the regulatory function, we focused on two allosteric regions in JAK1 JH2, the ATP-binding pocket and the αC-helix. Mutating L633 at the αC reduced basal and cytokine induced activation of STAT in both JAK1 wild-type (WT) and constitutively activated mutant backgrounds. Moreover, biochemical characterization and comparison of JH2s let us depict differences in the JH2 ATP-binding and strengthen the hypothesis that de-stabilization of the domain disturbs the regulatory JH1-JH2 interaction. Collectively, our results bring mechanistic understanding about the function of JAK1 in different receptor complexes that likely have relevance for the design of specific JAK modulators.  Janus kinases  are non-receptor tyrosine kinases that play a critical role in cell signaling via type I/II cytokines and interferons (IFNs) ,5. Some JAK1, as well as other JAK family members, harbor pathogenic gain-of-function mutations (GOFs) that cause acute lymphoid and -myeloid leukemia . JAK1 mutations are found in varying types of cancers; e.g., 9% of hepatocellular carcinoma patients have been found to have JAK1 mutations . In addiHomologous mutation to JAK2 V617F in JAK1 (V658F) causes acute lymphoblastic leukemia . In addiCurrently there are three JAK JH1-targeted inhibitors in clinical use, but recently targeting JAK JH2s with small molecular compounds has gained interest as a potential treatment for constitutively active cytokine signaling ,23,24. TAlthough the general principles of the JAK-STAT signaling are well established, the underlying differences between the function of JAKs and their individual domains in different cytokine signaling pathways are not fully defined. Moreover, the unbalanced number of JAKs (four) versus the cytokine pathways they transduce (over fifty) raises the question about the mechanisms that allow the versatile function of JAKs. For example, JAK1 is employed by the IL-2, IL-4, IL-10, and gp130  receptor families as well as type I and type II interferons . Intrigued by these questions, we set to study the role of JAK1 and particularly its JH2 in various signaling pathways. Our results show that JAK1 has varying roles in different receptor complexes, and that JH2 mediates important allosteric regulation of the JAK activity.To investigate the role of individual JAKs in heterodimeric receptor complexes, we focused on IL-2, IFNγ, and IFNα receptor systems. These pathways utilize JAK1 but consists of different JAK dimers: IL-2-signaling is driven by JAK1-JAK3, IFNγ by JAK1-JAK2, and IFNα by JAK1-TYK2. First, we studied how dependent the signaling systems are to the presence of an individual JAK. For this, we used cell lines that lack specific JAK expression. JAK1 and JAK3 deficient U4Cγβ-, JAK2 deficient γ2A- or TYK2 deficient 11.1 human fibroblast cells were transiently transfected with one or both JAKs relevant to the pathway, and the expression of JAKs and STATs was detected from the cell lysates. In addition, the phosphorylation of STAT1 (pSTAT1) and STAT5 (pSTAT5) was assayed. Of note, HA-STAT5 was transfected into the U4Cγβ cells, since endogenous STAT5 could not be detected with this method. All of the studied cytokine receptor pathways required the expression of two different wild-type (WT) JAKs for the cytokine-dependent activation of STAT . In IFNγNext, we studied the JAK dependency of the three signaling systems in the context of hyperactive mutations. JAK2 V617F mutant or homologous mutations in JAK1 and TYK2 see  were traIn contrast, the pathogenic JAK3 R657Q residing in the JH2-JH1 interface was strongly dependent on the presence of JAK1 WT, shown as abolished STAT5 activation when JAK1 was not transfected into the U4Cγβ cells  systems. In IL-2 signaling, JAK1 dominantly mediated the STAT5 phosphorylation and the activation requires both JH1 and JH2 domains. In the IFNγ and IFNα signaling, STAT1 activation requires both JAK1 and JAK2/TYK2, and IFNγ signaling shows to be dependent specifically on the pseudokinase domain of JAK1. We continued the analysis of JAK1 JH2 with available information from other JAK JH2s and focused on regions that have been identified to allosterically regulate JAK activation. We focused on the hydrophilic outer face of the JH2 αC and introduced a mutation in JAK1 that corresponds to JAK2 E592R A. JAK2 E72  couples the ATP phosphates to the αC-helix [αC-helix is an essential conserved region in protein kinases, and in the active kinase conformation the N-terminus of the αC-helix typically interacts with the activation loop phosphate. Furthermore, the conserved β3 LysαC-helix . The cliαC-helix  togetherαC-helix , 11 out To test the effect of the αC modulation in cell-based assays, JAK1 L633K was transfected into U4Cγβ cells with STAT1 or STAT5 responsive luciferase reporters and the normalized luciferase values were compared to cells transfected with JAK1 WT. L633K reduced the cytokine responsiveness in JAK1-JAK3 driven IL-2 signaling and JAK1-TYK2 driven IFNα signaling B. In addp < 0.001).Since JAK1 L633K reduced IL-2-driven STAT activation, we were interested whether the mutation can inhibit constitutively activated JAKs in cis, as shown with JAK2 . We creaNext, we set to compare the inhibitory potential between the αC-mutant and another allosteric region of JH2, namely the ATP-binding site. First, we showed that in addition to JAK2 I559F and JAK3 I535F mutations that have previously been shown to inhibit ATP binding and JAK hyperactivation, ,9 also hp = 0.12 between the basal vs. IL-2, 50 ng/mL). The pSTAT5 analysis of the mutant showed more variability, but also in this setting both the increased basal activity and the disturbed cytokine responsiveness were detected  C. The JATaken together, our cell-based and recombinant protein assays suggest that the JAK1 JH2 ATP-site differs from other JAKs in that the I597F cannot efficiently block ATP binding and shows increased basal activity and no inhibition with hyperactivating mutants. These unexpected effects to the signaling may be due to reduced stability of the mutated JH2 with I597F. This hypothesis is supported by data from JAK1 K622A that shows increased thermal stability and reduced signaling, probably stemming from the stabilizing interaction towards the αC that locks the whole domain in an inhibitory position.p-value 0.06). Similarly, large variation was detected also in IL-2 system with homologous JAK1 mutant although in the IL-2 system a distinct inhibition was observed . The JAK1 L633K was first transfected with active JAK3 R657Q into U4Cγβ cells and the pSTAT5 as well as the activity of endogenous STAT5 were detected . Correlaobserved . In the IFNα system, JAK1 L633K did not show inhibition of the co-transfected TYK2 V678F , and viccat) [After establishing that JAK1 L633K in the αC strongly inhibits IL-2 signaling, and that JAK1 K622A at the ATP site also reduced hyperactivation in cis, we wanted to compare the inhibition potential between the two regulatory JH2 sites in trans. JAK1 JH2 ATP site mutant K622A reduced but did not abrogate hyperactivation in the JAK3 R657Q background A and thecat) , which cModulating JAK JH2s can reduce constitutively active JAK signaling, but unlike the kinase-dead mutations that target the JH1 active site and completely abolish the signaling, JH2 mutants tend to maintain the cytokine responsiveness ,9,17. ThTo be noted, the expression levels of the JAK3 mutants are slightly reduced in comparison to the JAK3 WT in the As suggested by Hammarén et al., the disruption of the dimerization interaction between JH2 domains can be mediated by the in the αC and mutants in this region may result in complete loss of signaling. The JH2 ATP-binding site, on the other hand, likely affects the kinase activity in cis via regulating JH1 and thus have a weaker inhibitory potential .JAK1 is the most widely employed JAK by a variety of cytokine receptors but the underlying molecular mechanisms of JAK1 regulation are still largely elusive. One interesting question is whether JAK1 function is conserved between the different receptor systems, and in this work, we investigated the role of JAK1 in IL-2, IFNγ, and IFNα signaling. JAK1 null mice die perinatally and the newborn mice display a strong reduction of thymocytes, highlighting its importance in immunological and hematological functions . AccordiThe focus of our study was on the JAK1 pseudokinase (JH2) domain, where we compared its function in IL-2, IFNγ, and IFNα signaling. Our results show that in the tetrameric IFNγR-complex both JAK1 and JAK2 are required for STAT1 activation while in IL-2 signaling JAK1 functions plays a dominant role in STAT phosphorylation. Interestingly, in IL-2 signaling both JH1 and JH2 in JAK1 were indispensable for signaling while the JH2 domain in JAK1 was crucial for IFNγ induced STAT1 activation, and deletion of JH1 only reduced stimulation . Thus, oAlthough the structures of the JAK1, JAK2, and TYK2 FERM-SH2 domains have been solved with erythropoietin receptor (EPOR), leptin receptor (LEPR), and interferon receptors ,42,43, tIndividual JAKs show high specificity to distinct cytokine receptors. However, the study of Koppikar et al. showed that under some circumstances, conserved JAK pairing can be circumvented. They observed that long-term treatment with ruxolitinib results in resistance of JAK2 hyperactivation (persistence) that is caused by transphophorylation of JAK2 by JAK1 or TYK2 which allows constitutive STAT5 activation typically driven by homodimeric JAK2 . RuxolitRecently, Tvorogov et al. showed that ruxolitinib induces dose-dependent pJAK2, which can cause life-threatening cytokine-rebound syndrome (due to re-activation of STAT) when the drug is withdrawn . Again, In line with previous studies in IL-2 signaling, we showed that JAK1 is dominant over JAK3, and JAK3 is incapable of inducing STAT activation in the absence of JAK1 ,26. Our The apo structure of JAK1 JH2 has been solved but its regulatory function has not been investigated in detail. We focused on the allosteric JAK1 JH2 regions and their role in different signaling pathways and found that mutating residue L633 in the solvent-exposed face of αC-helix inhibits JAK1-driven signaling. The L633K mutant resides in the regulative JH1-JH2 interface and recently, a homologous JAK2 E592R mutant was shown to inhibit constitutive signaling in EPOR and IFNγR systems ,50. The Lastly, we introduced two mutations to disrupt ATP binding to JAK1 JH2. The JAK1 I597F mutation, homologs of which were shown to inhibit constitutive activation in JAK2 and JAK3 and TYK2, did not inhibit JAK1, JAK2, or JAK3 driven hyperactivation, but even increased the basal STAT5 activation. Interestingly, the mutation decreased the stability of the JH2 recombinant protein, and did not inhibit ATP binding. Another JAK1 JH2 ATP-site mutant, K622A inhibited STAT activation in both WT and JAK1 V658F context, and biochemical studies showed that the mutant stabilizes the JH2. To be noted, all tested JH2 mutants differed from the kinase-dead (JH1) mutants in that they maintained the cytokine inductivity. Combining the cell-based and recombinant protein derived data support mechanism of regulation where stabilizing JH2 inhibits hyperactivation while de-stabilizing further activates JAKs.Taken together, we have obtained detailed information of the JAK1 JH2 and identified differences in JH2 function in different cytokine receptor pathways. These results underline the importance of thorough understand of the mechanism of JAK signaling as a means to create safer and more efficient inhibitors. Full-length human JAKs were previously cloned in pCIneo expression vector by using SalI-NotI restriction sites. Full-length human STAT5A was in pXM vector. JAKs and STAT5A were C-terminally HA tagged. Site-directed mutagenesis was performed with QuikChange , according to the manufacturer’s instructions, and verified by using Sanger sequencing  supplemented with 10% FBS , 2 mmol/L L-glutamine (Lonza), and antibiotics . For transfection, cells were seeded on 24- or 96-well tissue-culture plates and transfected with FuGENE HD , according to the manufacturer’s instructions. Cells were transfected for 48 h and, where needed, cytokine stimulated in starvation medium without FBS for 15 min (for immunoblotting) or 5 h (for reporter assays), unless otherwise specified, with human recombinant IL-2 , IFNγ (PeproTech) or IFNα (PeproTech).Human fibroblast cell lines U4Cγβ, γ2A, and 11.1  were transiently transfected using Fugene HD reagent with different human JAK-hemagglutinin (HA) constructs in pCIneo vector (75 ng for each per 24 well plate well). If two JAK constructs were transfected simultaneously, equal amounts of DNA were used. If STAT5 phosphorylation was studied, 2 ng or HA-tagged STAT5 was co-transfected. After 48 h transfection, cell were stimulated (if needed) for 20 min and washed with cold phosphate buffered saline (PBS). Triton X-100 lysis buffer with protease and phosphatase inhibitors  was used to lyse the cells. Whole cell lysates were spun for 20 min at 16,000 g at 4 °C, and the resulting supernatants were run on 4–15% Mini-PROTEAN® TGX™ Precast Gels . Immunoblots were blocked with bovine serum albumin (BSA) and incubated with primary antibodies for HA Tag , phosphorylated STAT1 , STAT1 , or phosphorylated STAT5 , and with a mixture of goat anti-rabbit and goat anti-mouse DyLight secondary antibodies . Blots were scanned with an Odyssey CLx , and immunoblot signals were quantified with Image Studio software (LI-COR Biosciences) by manually assigning bands and dividing the phosphorylation (pSTAT1 or pSTAT5) signal values with the expression (STAT1 or HA) signals.STAT5 transcriptional activity was assessed by measuring the luciferase expression SPI-Luc 2) driven by a STAT5 responsive promoter, as described previously  driven b. U4Cγβ, JAK1 JH2 constructs spanning residues 561–852 of human JAK1, of the wild-type sequence or either I597F or K622A mutations were sub-cloned into pFastBac vector for expression as N-terminal glutathione-S-transferase (GST) fusion proteins with a tobacco etch virus (TEV) proteins. Constructs were expressed in High Five insect cells (Thermo Fisher Scientific) using the Bac-to-Bac baculovirus expression system  according to manufacturer’s instructions. After protein expression , the cells were collected by centrifugation, resuspended in lysis buffer containing 50 mM Tris HCl pH 8.0, 10% Glycerol, 500 nM NaCl, 1 mM TCEP supplemented with phosphatase and protease inhibitors , and lysed by applying four freeze-thaw cycles. The lysates were clarified by centrifugation and incubated 1.5 h with GSH-coupled resin beads (Protino Glutathione Agarose 4B). Beads were washed and the protein detached by digesting with a tobacco etch virus (TEV) protease (overnight at 4 °C). The flow-through was concentrated and run on a Superdex 200 gel filtration column equilibrated in final buffer . The eluted peak was concentrated and stored at −80 °C.Thermal-shift experiments were carried out in 96-well PCR plates in a final volume of 25 μL with the following reagent concentrations: 6x Sypro Orange , 3 μM protein Ni-NTA eluate, 1 mM MgCl2, 20 mM Tris pH 8.0, 500 mM NaCl, 10% glycerol, 4 mM DTT and 0.02% CHAPS, and 1 mM ATP. Reactions were heated in a real-time CFX96 PCR cycler (Bio-Rad) at 1 °C per min from 4 °C to 95 °C with a fluorescence read every 1 °C. Fluorescence data were then normalized to represent unfolding curves, which were fitted to a Boltzmann sigmoidal equation with GraphPad Prism to obtain average Tm values with errors as SD.Our studies show that JAK1 has varying roles in IL-2, IFNα, and IFNγ systems. Our results demonstrate that the pseudokinase domain (JH2) of JAK1 is an important regulatory region and modulating it can either down, or up-regulate JAK1-driven signaling. Specifically the outer face of the JH2 αC-helix was effective in reducing wild-type and constitutive STAT activation, although the potency dependent on the JAK and the related signaling complex. Furthermore, we highlighted biochemical and biological differences between the ATP-binding sites of JAK JH2s, which can be beneficial in design novel JAK modulators targeting outside the conserved kinase domain.


Uric acid was the strongest predictor of PWV and was associated with the highest risk for increased AS. The interaction analysis showed that the joint effect of increased UA and HOMA was significantly higher than that expected in the absence of interaction under the additive model, indicating that the two biomarkers synergically interacted for promoting vascular damage. Our data showed that UA interacted with IR to increase AS in a large cohort of newly diagnosed, never-treated hypertensive patients. Increased levels of uric acid (UA) have been shown to be correlated with many clinical conditions. Uric acid may adversely affect the insulin signalling pathway inducing insulin resistance (IR). Several studies report the association between arterial stiffness (AS), an early indicator of atherosclerosis, and UA. The purpose of the present study was to evaluate the association between UA and AS, considering the potential role of IR. We enrolled 1114 newly diagnosed, never-treated hypertensive patients. Insulin resistance was assessed by the homeostatic model assessment (HOMA) index. Arterial stiffness was evaluated as the measurement of the carotid–femoral pulse wave velocity (PWV). The study cohort was divided into subgroups, according to increasing tertiles of UA. The mean values of UA were 5.2 ± 1.6 mg/dL in the overall population. Pulse wave velocity was linearly correlated with UA ( Uric acid (UA) is mainly synthesized by the hepatic tissue as the end product of purine catabolism and excreted by the kidney; it circulates predominantly in the form of a monovalent sodium salt (urate) . Hyperur2. None had a positive history or clinical evidence for myocardial infarction, angina, valvular heart disease, hypercholesterolemia, diabetes mellitus, coagulopathies, peripheral vascular disease, or diseases predisposing to Raynaud’s phenomenon or vasculitis. Additional exclusion criteria were post-menopause or hormonal substitutive therapy for women, and positive anamnesis for drug or alcohol abuse, gout, and/or treatment with allopurinol and/or other drugs that may affect UA metabolism. Secondary forms of hypertension were excluded by systematic testing following standard clinical procedures, including measurement of aldosterone and plasma renin activity, Doppler studies of the renal arteries, and/or renal angiography or renal scintigraphy. We recruited 1114  newly diagnosed hypertensive, never-treated, non-diabetic Caucasian individuals participating in the Catanzaro Metabolic Risk factors (CATAMERI) Study [Measurements of clinic blood pressure (BP) were performed after 5 min of rest in the left arm of the supine subjects with an aneroid sphygmomanometer. A minimum three BP records were considered on three different measurements at least two weeks apart. Systolic and diastolic BPs were registered at the first appearance (phase I) and the disappearance (phase V) of Korotkoff tone. Baseline values of BP were calculated from the mean of the last two of the three consecutive recordings performed at three minutes intervals. Subjects with a systolic BP (SBP) > 140 mmHg and/or a diastolic BP (DBP) > 90 mmHg were considered hypertensive in accordance with current guidelines . All laboratory measurements were performed following a 12 h minimum fasting period. Plasma glucose concentrations were measured by the method of glucose oxidation . Triglycerides, total, high- (HDL) and low-density lipoprotein (LDL) cholesterol concentrations were evaluated by enzymatic methods . Concentration of plasma insulin was assessed by a chemiluminescence-based assay (Roche Diagnostics). Insulin sensitivity was calculated with the homeostasis model assessment (HOMA) index, computed using the following formula: (fasting insulin (µU/mL) × fasting glucose (mmol/L))/22.5. Serum creatinine was assessed using the Roche Creatinine Plus assay  on a clinical chemistry analyser . Renal function, evaluated by e-GFR, was calculated according to the equation suggested by the Chronic Kidney Disease Epidemiology (CKD-EPI) Collaboration group . Serum UAll measurements were conducted using a widely validated system  based on high-fidelity applanation tonometry (Millar) and appropriate software for the analysis of pressure waves (Sphygmocor™), as previously reported . After aBaseline characteristics are reported as mean ± SD for continuous variables and frequencies and percentages for categorical data. One-way ANOVA and chi-square test were used to test for differences among tertiles of UA concentrations for categorical values and continuous variables, respectively. Linear regression analysis was employed to assess the correlation of PWV with the following covariates: age, BMI, SBP, DBP, HDL and LDL-cholesterol, triglycerides, high sensitivity C reactive protein (hs-CRP), e-GFR, HOMA and serum UA concentrations. Afterwards, variables reaching statistical significance were inserted in a stepwise multivariate linear regression model to define the independent predictors of PWV, AI and AP. Furthermore, HOMA only, and not fasting insulin or glucose levels, was included in the model to avoid possible collinearity.Finally, a linear regression analysis was performed to test the association between traditional CV risk factors and serum UA with increased arterial stiffness (AS) considered as PWV >10 m/s . Uric acThe investigation of biological interactions between UA and HOMA that may explain increased stiffness was performed, as previously described , by divip < 0.05. All analyses were carried out with the statistical package SPSS 20.0 for Windows .Differences were assumed to be significant for All evaluations were made according to the Declaration of Helsinki after obtaining approval by the local Ethical Committee and written informed consent from each subject (code protocol number 2012.63).p = 0.022).Peripheral and aortic hemodynamic parameters of the whole study population and of the three subgroups are shown in r = 0.453, p < 0.0001), HOMA , hs-CRP , SBP , age  and LDL , while HDL values and UA levels showed an inverse relationship . The correlation between PWV and several covariates in the whole study population was assessed by linear regression analysis . Pulse wTo identify the independent predictors of PWV, variables showing statistically significant correlations in the linear regression analysis were included in a stepwise multivariate linear regression model, with gender and smoking status as dichotomic values . Serum UA was the strongest predictor of PWV, justifying 20.5% of its variation. Other predictors were HOMA, SBP, e-GFR, age and hs-CRP, accounting for another 6.3%, 0.9%, 0.7%, 0.5% and 0.3% of its variation, respectively. Finally, in the adjusted logistic regression analysis, a 1 mg/dL increase in serum UA values resulted in an 80% increased risk of AS, defined as PWV > 10 m/s (p < 0.0001).In the univariate logistic regression analysis, serum UA  and HOMA  values above the median were both associated with a greater risk of increased AS (PWV > 10 m/s).A biological interaction analysis performed adjusting for a series of potential confounding factors  highlighted a significant interaction between UA and HOMA for increased AS risk. Specifically, the interaction analysis carried out according to an additive model, which represents the preferred model for testing a biological interaction in etiological studies ,22, showNotably, when the interaction analysis was conducted according to a multiplicative model, both on univariate and multivariate logistic regression models, the OR observed for vascular damage in subjects with both serum UA and HOMA values above the median was greater than the simple product between the OR in patients with serum UA or HOMA values above the median  .Several previous studies have provided evidence suggesting a possible correlation between subclinical organ damage and increased serum UA ,25,26,27Our data showed that patients in the highest UA tertile (mean UA serum levels 7.1 ± 1.1 mg/dL) had a worse metabolic and hemodynamic status with significantly higher levels of PWV and its hemodynamic correlates AI and AP. A further proof for the role of UA levels in vascular damage was obtained from the correlational analysis, demonstrating that UA was the major predictor of PWV justifying 20.5% of its variation. Interestingly, from the adjusted logistic regression analysis emerges that an increase of 1 mg/dL in serum UA values results in an almost double risk of AS, defined as PWV > 10 m/s. This observation has an important clinical relevance, because it is demonstrated that AS is an independent predictor of cardiovascular events and mortality from all-cause. Specifically, an increase of 1 m/s of PWV raises by 14% the global risk of CV events, by 15% the risk of cardiovascular mortality, and by another 15% the risk of all-cause mortality . The resFinally, our study offers, to the best of our knowledge, the first proof of a biological interaction between UA and HOMA index in increasing the risk of AS. Particularly, patients with both UA and HOMA values higher than the median value presented a six times greater risk of PWV > 10 m/s. This finding was observed both in male then in female patients, but the effect was more pronounced in males. This demonstrates a synergistic action between UA and HOMA in inducing subclinical vascular damage, suggesting that IR itself may be one of the major mechanisms trough which UA increases AS in our cohort of newly diagnosed, never-treated hypertensive individuals. The ability, demonstrated in experimental studies, of UA to interfere with insulin signalling , with IGThe present study has a number of strengths including the size of the cohort analysed, the exclusion of confounding conditions known to affect UA levels, the recruitment of newly diagnosed subjects naive to drugs that may alter UA concentration and the inclusion of childbearing-age women only to account for the established effect of the loss of oestrogen uricosuric action. Nonetheless, the cross-sectional design of our study prevents the demonstration of a causal relationship between higher levels of UA and increased AS, even if previous preclinical ,33 and cFurthermore, the present data have been obtained in Caucasian individuals, whether our findings can be extended to subjects from different ethnic groups remains to be determined. Finally, another important limitation of the study consists in the fact that we have not measured physical activity at baseline, and it is well known that it represents an important factor affecting AS.In conclusion, the results emerging from our study strengthen the available evidence on the importance of UA as a cause and predictor of cardiovascular damage. Furthermore, our data appear highly clinically relevant since the knowledge of the pathophysiological mechanisms may allow early interventions on vascular damage to prevent clinical events.