After an initial ankle sprain, a relevant number of participants develop chronic ankle instability (CAI). Compensatory strategies in patients with CAI may change the inter-limb symmetry needed for absorbing movement-related forces. Accordingly, an increased risk of injury can occur. The present study aimed to compare the inter-limb asymmetry of kinetic and electromyography between individuals with CAI and without a history of an ankle sprain (Non-CAI) during walking. In this cross-sectional study, fifty-six athletes  participated. Participants walked at a comfortable pace over level ground while vertical ground reaction force (vGRF) and muscle activity of the tibialis anterior, peroneus longus, medial gastrocnemius, and gluteus medius were recorded. Inter-limb asymmetry during walking was calculated for each of the variables. Patients with CAI exhibited a greater inter-limb asymmetry of the first peak of vGRF, time to peak vGRF, and loading rate (P < 0.001), as well as presenting a greater inter-limb asymmetry of peroneus longus activity (contact phase) (P = 0.003) and gluteus medius activity (midstance/propulsion phase) (P = 0.010) compared to the Non-CAI group. No other differences in vGRF or muscles activity were observed between the groups. Our findings indicate that patients with CAI walk with greater inter-limb asymmetry in vGRF and muscle activity in different phases of the gait cycle compared to Non-CAI group. Our results could inform future studies on gait training aimed to reduce asymmetry during walking in patients with CAI. Lateral ankle sprain, in particular, is a common musculoskeletal injury that is often found among adolescent athletes3. After an initial ankle sprain, recurrent sprains, subjective feelings of instability, and disabling symptoms such as pain and swelling continue to affect 40% of patients at least one-year post-injury4. Persistent “giving way” episodes and recurrent injury are the obvious features of a heterogeneous condition known as chronic ankle instability (CAI)5. Residual and persistent symptoms associated with CAI can lead to decreased physical activity7, reduced quality of life8, and an increased risk of ankle osteoarthritis after injury9. Both lateral ankle sprains and CAI have been linked to decreased cartilage health and post-traumatic osteoarthritis10, accounting for 78% of end-stage ankle osteoarthritis cases10.The ankle joint is a primary sublocation for injury in athletics11, impairing the ability to minimize joint loading and protect the joint against impairments12. Increased plantar flexion moment and impact ground reaction force in patients with CAI during initial contact of walking increase the load at the ankle joint in the sagittal plane13. It has been hypothesized that alterations in reflexive, sensory, and motor control strategies contribute to CAI5. For example, during walking, patients with CAI demonstrate strategies of neuromuscular-activation across their lower extremities, which were different from strategies presented by those Non-CAI16. However, the muscle activity profiles of patients with CAI during walking are conflicting. For example, patients with CAI have been reported to have increased peroneus longus and tibialis anterior activation during the early stance of walking17. Similar results have been reported by Delahunt et al.18 and Louwerens et al.19 concerning increased peroneus longus and tibialis anterior activation, respectively. On the other hand, some other studies22 have reported a lack of difference between CAI and Non-CAI groups. Further, research has also reported a significant decrease of tibialis anterior, peroneus longus, and gluteus medius activity during walking of patients with CAI13. It should be noted that these studies used different reference tasks for EMG normalization22, which complicates their direct comparison.Initial lateral ankle sprains and subsequent sprains  damage the sensorimotor system and can alter the dynamic restraint mechanisms of the lower extremity15 and more intense physical activities such as running23. Specifically, patients with CAI had higher vertical ground reaction force (vGRF)25 and vGRF loading rates24, relative to Non-CAI individuals during early stance of walking. The vGRF and vGRF loading rate play a role in cartilage health28. Higher loading rates cause more chondrocyte death and cartilage matrix damage28. Patients with CAI experience changes in matrix densities as early as one year following initial injury29 and are at increased risk for joint degeneration and post-traumatic osteoarthritis30, indicating less cartilage resiliency and elasticity against repetitive load31. The relationship between altered loads and ankle articular cartilage health has not been assessed in patients with CAI up to now. However, the alteration of walking biomechanics 32) observed in patients with CAI might affect articular cartilage metabolism and health, thus increasing mechanical stress on a specific portion of the ankle cartilage. For example, Hintermann et al.33 showed that patients with CAI have 62% of ankle cartilage lesions in the talus's medial areas. It has also been reported that degenerative arthritis of the ankle has mainly occurred due to unbalanced mechanical loading in the medial ankle joint of CAI patients34. Therefore, quantification of vGRF differences in individuals with CAI compared to Non-CAI is warranted as a precursor to assessing the relationship between load and cartilage health in this population. Understanding differences in how patients with CAI load their ankles may assist with developing the treatment to slow ankle joint degeneration.Patients with CAI have presented altered kinetics during walking35, which increases the risk of contralateral limb injury37. Eighty-five percent of people who experience CAI after ankle sprains also have problems with the contralateral limb38. This inefficiency may contribute to the high recurrence rates in patients with CAI38. One possible way that can help to understand the mechanism of limb coordination in walking is to study inter-limb asymmetry. Investigating the asymmetry in patients with CAI can provide insights into the control of walking and the importance of bilateral deficits in the occurrence of injury. This finding may be unique from more conventional measures such as velocity and may guide the clinicians’ treatment decisions. Previous research emphasises optimal balance in lower limb symmetry of healthy individuals39. However, in limb-synchronous movement tasks such as walking, compensatory strategies may disturb the inter-limb symmetry necessary for absorbing movement-related forces, potentially increasing the risk of injury40.Frequently, ankle injuries cause bilateral defects, as patients compensate to protect the injured joint41 and muscle activity16 during walking in patients with CAI. Also, previous studies have not collected information about uninjured limb for CAI patients. Patients with CAI could exhibit biomechanical compensatory strategies to the contralateral limb but cannot be observed43. However, evaluating inter-limb asymmetry of vGRF and muscles activity in patients with CAI is essential, which can provide more complete information in these patients. Many of the features can be obtained from GRF and muscle activity to distinguish between normal and abnormal patterns of motor behavior. Thus, the study of the symmetry of GRF and electromyography variables in patients with CAI represents an opportunity because it is a less investigated area. Therefore, the present study aimed to compare the inter-limb asymmetry of vGRF components and selected lower limb muscles activity between individuals with CAI and Non-CAI during walking. We hypothesized that patients with CAI will exhibit greater inter-limb asymmetry in vGRF components and muscles activity compared with individuals without CAI during walking.There is limited research evaluating the compensatory strategies utilized by the sensorimotor system in patients with CAI, and the effect these may have on inter-limb symmetry. Additionally, previous research has reported mixed results regarding vGRFThis study incorporated a single-session cross-sectional design that was conducted at the Biomechanics Laboratory of Kharazmi University. The independent variable was group . The dependent variables were inter-limb asymmetry in vGRF components  and muscle activity . Prior to participation in the study, all subjects were briefed about the objectives and provided written informed consent, and all participants provided written informed consent prior to enrollment. This study was performed following the 1964 Helsinki declaration, its later amendments and local ethics committee by the Research Ethics Committee of the Sport Science Research Institute (IR.SSRI.REC.1400.1160).44. Namely, inclusion criteria for the CAI group were: (1) having at least one significant ankle sprain, with the initial sprain occurring at least 12 months prior to study enrolment; (2) having sustained the most recent ankle sprain more than three months prior to enrolment; (3) having a history of ankle “giving way” or feeling of instability, which occurred at least two episodes in the last six months; and (4) reporting a score < 24 on the Cumberland Ankle Instability Tool (CAIT), < 90% on the Foot and Ankle Ability Measure (FAAM)–Activities of Daily Living (ADL) subscale and < 80% on the FAAM–Sport subscale. The Non-CAI group included healthy participants with no acute symptoms of an ankle injury and no history of ankle sprain or symptoms of instability. Exclusion criteria for both groups were: (1) having a history of previous surgeries to the musculoskeletal structures in either limb of the lower extremity; (2) having a history of fracture in either limb of the lower extremity requiring realignment; and (3) acute injury to musculoskeletal structures of other joints of the lower extremity in the previous three months. All athletes have right lower limb dominance. Also, only athletes with unilateral right ankle CAI were enrolled. Limb dominance was defined as the preferred limb to kicking a soccer ball45.Eligibility criteria for the CAI group were based on the standards approved by the International Ankle ConsortiumPatients of the CAI group were matched on age and gender with individuals of the Non-CAI group. One hundred and twenty volleyball and basketball athletes were screened. For this study, we defined an athlete as a person having at least two training days/week and one match/week (in-season). The participants were classified into CAI and Non-CAI groups. Determination of the sample size was based on data from a pilot study that was calculated according to the inter-limb asymmetry of vGRF, a power analysis was conducted using G*Power version 3.1. The analysis revealed that given the effect size of 0.80, at least 21 participants per group was needed for a power of 0.80 and an alpha level of 0.05. Due to potential attritions, 56 athletes met the inclusion criteria (28 per group) and were recruited and tested for this study.The vGRF data were collected at a sampling rate of 1000 Hz with Two force plates  embedded into a 8-m walkway (approximately 4.5-m prior to force plate contact). Force plates were positioned respectively with 17 cm and 60 cm center to center distance in medial–lateral and anterior–posterior directions.8 Ω input impedance, 108 dB  CMRR, 32 kHz bandwidth and gain of 4000 ×). Signals were measured using disposable electrodes  with a diameter of 10 mm. The EMG system was synchronized to the force plates.Electromyographic (EMG) data were recorded using EMG system  applied over the tibialis anterior, peroneus longus, medial gastrocnemius, and gluteus medius. These muscles were selected because of their role in controlling the movement of the frontal and sagittal planes during walking. EMG signals were recorded at a sampling frequency of 1000 Hz and differential pre-amplified circuit . For vGRF, two peaks were extracted: the maximum of the first half (first peak or impact peak) and the maximum of the last half (second peak) during the stance phase. Time to peak vGRF was defined as the time from initial heel contact to the first peak of vGRF24. The loading rate was defined as the rise in force (N/BW) from heel contact to the first peak of vGRF, divided by time to the first peak of vGRF24. These vGRF components were extracted for both limbs in both CAI and Non-CAI groups.The vGRF data were filtered using a fourth-order, zero-phase lag, low-pass Butterworth filter with a cut-off frequency of 15 Hz45.The speed of walking was measured as the length of step as divided by the duration of step, e.g., distance between the center of pressure (COP) anterior–posterior position at successive heel contact on each force plate (considering the 60 cm between them) divided by the time interval between those successive contacts on the force plates, took as the number of force samples divided by sampling frequency43. In order to analyze the measured data, the stance phase of gait cycles was identified based on vGRF data (with a threshold set at 10 Newtons) and were normalized from 0 to 100%. Root mean square (RMS) activity index was used to calculate the amount of electromyography activity of the muscles during walking, during the contact phase (the first 25% of the stance phase), and the combined midstance/propulsion phase (the latest 75% of the stance phase). Analyses were performed on the RMS calculated with a moving window of 50 ms width. The RMS was normalized to the maximum voluntary isometric contraction (MVC) of each muscle and are reported as a percentage of the MVC (%MVC). The MVC was obtained during maximum contraction tests with manual muscle tests. The maximal value of MVC testing was collected over three trials for each muscle. A one-minute rest period was given between the MVC trials. All data processing occurred in MATLAB software .The raw EMG data were filtered with a fourth-order, zero-phase lag, bandpass Butterworth filter with a cut-off frequency of 10–450 Hz50: Asymmetry Index = (injured (or dominant) limb − uninjured (or non-dominant) limb) × 2/ (injured (or dominant) limb + uninjured (or non-dominant) limb) × 100%. Therefore, a negative asymmetry would be indicative of a higher value for the uninjured (or non-dominant) limb. Asymmetry of less than 10% can be considered acceptable49.The inter-limb asymmetry for the variables of vGRF or muscles activity was calculated using an equation from previous studiesWe used the Shapiro–Wilk test to determine the normality of the data (p < 0.05). Independent samples t-tests were computed to compare participant demographics, CAIT, FAAM-ADL, FAAM-Sport scores and the primary dependent variables between CAI and Non-CAI groups. In addition to inferential statistics, effect sizes (ES) (bias corrected Hedges’ g) and 95% confidence interval (CI) were also computed to estimate the precision and magnitude of group differences, given the multiple t-tests ran. ES were interpreted as weak (≤ 0.40), moderate (0.4 –0.69), or strong (≥ 0.70). Also, to compare the vGRF components and muscle activity between the CAI and Non-CAI groups, a two-way ANOVA was conducted to assess the interactions of group (CAI and Non-CAI) and limb (injured/dominant and uninjured/non-dominant). The injured and uninjured limbs of the CAI group were matched with the dominant and non-dominant limbs of the Non-CAI group, respectively. When significant differences were observed, pairwise post-hoc comparisons were performed using the Bonferroni test for multiple comparisons. All statistical analyses were computed using SPSS software, version 22 and statistical significance was considered when p < 0.05.The demographic characteristics of the participants are summarized in Table Patients with CAI demonstrated more asymmetry in the first peak of the vGRF , time to peak vGRF , and loading rate   , time to peak force , and rate of loading . The post-hoc Bonferroni test showed significantly greater magnitude of the first peak of the vGRF (P < 0.001), time to peak vGRF (P = 0.002), and loading rate (P = 0.016) in the injured limb of patients with CAI compared to the dominant limb of the Non-CAI group  , and gluteus medius activity (midstance/propulsion phase) . The Bonferroni test showed significantly greater peroneus longus (P = 0.004) and gluteus medius activity (P = 0.011) for the injured limb of patients with CAI compared to the dominant limb of the Non-CAI group  and gluteus medius activity (at the midstance/propulsion phase). The asymmetry observed in the vGRF components and muscles activity of the CAI group is due to changes in the injured limb. However, the results of this study did not show a significant difference between the uninjured limb of the CAI group and the non-dominant limb of the Non-CAI group in any of the variables.2. Therefore, preventive initiatives are necessary to reduce the long-term consequences of these injuries51, and the first step towards progress in this area is to examine the walking features of these patients.Investigating inter-limb asymmetry can offer more comprehensive information to explain injured and uninjured limbs in the stance phase of walking. However, using the injured limb data alone may lead to unclear results, especially in athletes with CAI who may have to adapt to maintaining ankle stability. Also, our study tried to focus on unilateral right ankle CAI, gender, athlete performance, and the number of athletes in each sport to analyze a more homogeneous sample. Potentially, the most worrying long-term outcome of CAI is the development of post-traumatic osteoarthritis52. Previous studies confirm that patients with CAI experience different magnitudes of vGRF compared to the Non-CAI group during running23, jump landing52, and cutting53. However, few studies reported vGRF data in patients with CAI during walking41. Read et al.25 and Son et al.13 noted increased vGRF in patients with CAI than the Non-CAI group during early stance of walking. Contrary to our findings, Moisan et al. in their published abstract, reported no significant differences in vGRF between individuals in the CAI and Non-CAI groups41. Moreover, Wikstrom et al.24 reported that patients with CAI had higher loading rates and less time to peak vGRF compared to the Non-CAI group during walking. However, they reported no differences between groups in magnitude of vGRF during overground walking at a self-selected pace24. This difference between Wikstrom et al. and our study is due to the point that they normalized the vGRF to body weight and gait velocity, while in our study it was only body weight.Patients with CAI demonstrated inter-limb asymmetry in the first peak of vGRF, time to peak vGRF and loading rate compared to the Non-CAI group. The primary finding is that patients with CAI in the injured limb had a significantly higher first peak of vGRF and loading rates and significantly less time to peak vGRF than the non-dominant limb of the Non-CAI group, consistent with broader biomechanical literature on CAI32, increased plantar flexion moment13 and increased loading rates at initial contact24 in comparison to the Non-CAI group. Less dorsiflexion or more plantar flexion during the early stance of walking may reduce the ability to absorb impact at heel strike. Cumulatively, these findings would suggest that patients with CAI have a limited ability to appropriately modulate vGRF during walking. The primary analyses also show that the CAI cohort has problems in dampening forces during gait termination54, indicating limitations in force modulation across gait-related activities.Patients with CAI may compensate for ankle instability with a more rigid walking strategy as a mechanism to protect the ankle joint from recurrent sprains. For example, patients with CAI have decreased dorsiflexion angle or increased plantar flexion52, the present results are likely to be a neuromuscular response that may be attributed to sensorimotor impairments that increases ankle joint stability during early stance as a protective mechanism. However, they may contribute to post-traumatic ankle osteoarthritis. Patients with post-traumatic ankle osteoarthritis also show changes in peak force and loading rates during dynamic activities such as stair climbing55 and walking56. Additionally, it has been shown that articular cartilage catabolism changes relative to alterations in BW and GRF because of walking57. We speculate that these data points can be linked by extrapolating the increased force across the number of steps per day over years. Therefore, changes observed in the loading pattern may increase wear on the talar cartilage and aid in the progression of post-traumatic osteoarthritis. This theoretical model is supported by previous research in patients with other musculoskeletal injuries. For example, increased vGRF was associated with biochemical markers of cartilage degradation in patients with anterior cruciate ligament reconstruction (ACLR)59. Despite preliminary evidence from related studies in ACLR literature, the relationship between cartilage degradation and increased peak force and loading rates during walking has not been explored in CAI populations. However, only a prospective research design will be able to test this hypothesis adequately.Although the increased vertical forces in the present study are considered to be the underlying causes of musculoskeletal stress injuries and overuse injuries18 in patients with CAI. However, the results from our muscle activation during walking should be interpreted cautiously. For example, our results are not in line with researchers who reported a decrease in peroneus longus during the initial stance60, or lack of difference between the groups43. The variations in the reference task adopted for EMG normalization by different researchers induce a problematic comparison of results across studies. For example, the reference task was MVC in this study, while others used squatting positions13 or quiet standing60. Thus, knowing if the groups utilized more or less motor activity while doing those normalization tasks represents a priority for future research.This study showed significantly greater peroneus longus asymmetry in the contact phase that did not continue into the midstance/propulsion phase participants in the CAI versus the Non-CAI groups. Also, our findings show that patients with CAI in the injured limb had significantly higher peroneus longus activity relative to the dominant limb of the Non-CAI group. This result agrees with previous investigations demonstrating greater activity of the peroneus longus muscle during early stance18 and plantar flexion13, as well as an increased lateral COP displacement60 during walking in patients with CAI compared to the Non-CAI group. These differences are risk factors for lateral ankle sprains. In addition, it is likely that the abnormal peroneus longus activation is a strategy to compensate for neural alterations affecting ankle evertor muscles61. This increased duration and magnitude of peroneus longus activity in patients with CAI contrasts that of healthy individuals who activate the peroneus longus during midstance to help initiate pronation and stabilize their first ray during propulsion phases of gait62.The higher activation of the peroneus longus could be a compensatory strategy to avoid ankle inversion movements leading to ankle sprains, since a number of studies reported an increased ankle inversion63. For example, Koldenhoven et al. reported that the gluteus medius muscle had higher activity before heel contact, during the second half of the stance phase, and the first quarter of the swing phase as compared to healthy participants42. They suggested that Proximal changes in gluteus medius muscle activity may represent a coping mechanism that CAI patients utilize to generate a wider base of support or stabilize their lower limb during walking42. Although Feger et al. did not find significant differences in EMG amplitude and onset of gluteus medius activity during walking, they reported earlier onset activity as well as overactivity just before and just after initial contact20. Contrary to our findings, Moisan et al.43 and Son et al.13 observed decreased gluteus medius activity during the stance phase. These differences in results may be due to the use of different methods in EMG normalization, as they used the peak RMS amplitude while walking43 and the squat position13 for normalization. Also, in the study of Moisan et al.43, participants used shoes while walking, and the whole stance phase was considered, while our participants were barefoot and we considered several stance phases. Accordingly, future studies are needed to investigate the gluteus medius muscle function in patients with CAI during walking.In the midstance/propulsion phase, the inter-limb asymmetry in the gluteus medius activity was significantly different between individuals in the CAI and Non-CAI groups. Also, our results indicate that patients with CAI in the injured limb had significantly higher gluteus medius activity than the dominant limb of the Non-CAI group. Modified movement control patterns in the muscles of the proximal joints have been identified in patients with CAI64. It has been argued that total-body balance in the frontal plane while walking is modulated by the subtalar and hip joints that control the center of mass position and upper extremity motion64; thus, the ankle and hip function synergically. When the foot enters a susceptible position in danger of lateral ankle sprain  in the frontal plane, wrong foot placement could be rectified by an interface between hip abductors and evertors, which shows the significance of gluteus medius function in CAI. Given that increased gluteus medius activation would decrease hip adduction  and create efficient balance of the upper extremity, which impedes lateral movement of center-of-mass, potentially stops patients with CAI to be inflicted by lateral ankle sprains. So, increased gluteus medius activation can be considered as a “safety strategy” to compensate for a changed compensatory motor control system. Continued increased functional load on the hip joint can increase the ground reaction shear forces.The gluteus medius muscle contributes to balance control of the upper extremity  in the frontal plane while the early stance and midstance phases of walking25. Increased abnormal stress on the osseous structures and soft tissues can cause osteoarthritis prematurely56. The collective aberrant kinetic and muscle activation patterns demonstrated by patients with CAI in this study suggest that therapeutic interventions should focus on restoring normal neuromuscular function during walking. Specifically, targeted interventions to restore vGRF symmetry, reduce peroneus longus asymmetry, and restore gluteus medius function may have important implications for the progression from CAI to ankle joint degeneration. Real-time biofeedback tools and injury prevention programs can modify vGRF66. Although there is no evidence-based recommendation for CAI gait training, it may support the rehabilitation of inter-limb asymmetry and reduce the risk of re-injury67. We surmise that for asymmetry changes to be maintained by patients with CAI, a more inclusive approach to gait training is probably needed to restore proper vGRF and improve the patterns of muscle activity as these factors play a role in an patient’s loading pattern.Asymmetry, occurring due to increased muscle activity and vGRF components in the injured limb in the CAI group, is a compensatory (coping) gait pattern that may allow patients to increase limb stability. The asymmetry created in the CAI group indicates altered gait mechanics, which could increase loads on lower extremity joints in the injured limb, thus potentially increasing the risk of re-injury42, other gait biomechanics studies on patients with CAI adopted functional statistics  to compare all data points across the gait cycle69, thus offering opportunities for future research in this field. Third, considering that participants in this study had a mean age of 23 years, our results can be generalized to only the targeted populations. Fourth, given the cross-sectional nature of this study, it is unclear whether the current CAI leads to the observed alterations or if gait asymmetry facilitated the development of CAI. Lastly, it is possible that the asymmetry is already present before the injury acting as a risk factor for injury/instability. The design of our research  prevent us from any definitive conclusion. Thus, future studies should investigate the topic using a prospective design .The present study had several limitations that should be mentioned. First, although the combination of vGRF and EMG was used in this study to evaluate the difference in asymmetry between the groups, kinematic evaluation of joint movements, which could contribute to the comprehensiveness of the study, was not performed in this study. Therefore, the interpretation of our results should be considered with some caution. Future research is needed to simultaneously examine EMG, vGRF and lower limb kinematics while walking to better understand the implications of our findings. Second, we compared two discrete time points throughout the gait cycle in our study. Although this analysis is recognized internationallyPatients with CAI walk with greater inter-limb asymmetry in muscle activity and vGRF in different phases of the gait cycle compared to the Non-CAI group. These motor behaviour changes could have occurred to maintain joint stability in patients with CAI. Our results could inform future studies on gait training aimed to reduce asymmetry during walking in patients with CAI.


Efficient pork production relies on a predictable supply of market pigs. Seasonal infertility caused by heat stress decreases fertility in sows during the summer months, impacting breeding targets and decreasing the efficiency of pork production. The present study examined the effect of a seminal additive containing caffeine, oxytocin, and lecirelin on the fertility and prolificacy of Iberian sows during two consecutive years. The results confirmed that inclusion of the additive in semen prior to AI decreased the seasonality effect, increasing the percentage of pregnant Iberian sows throughout the year.®, a commercial semen supplement demonstrated to increase fertility and litter size in commercial sows, was tested to improve reproductive performance in Iberian sows. A total of 1430 Iberian sows were artificially inseminated (AI) with semen from Duroc boars and assigned by parity to receive the seminal additive Suinfort® containing 2 IU oxytocin, 5 µg lecirelin, and 2 mM caffeine , or to serve as non-supplemented controls . CON showed a lower fertility comparing to winter for spring (p = 0.001) and summer (p < 0.001); summer was lower than autumn (p = 0.012). SF removed this seasonal effect (p > 0.05). Fertility was significantly higher for SF sows during summer (p = 0.025) and autumn (p = 0.004). Total born, live-born, stillborn, and mummified piglets did not differ between CON and SF but were impacted by the season, with total and live-born decreasing in summer compared with autumn (p < 0.001) and winter (p = 0.005). In conclusion, seminal supplementation with Suinfort® improved the fertility of Iberian sows during periods of seasonal infertility.Suinfort The pig industry depends on artificial insemination (AI) with liquid semen doses from selected boars. This technique has increased efficiency and genetic improvement , yet it An additional challenge is the seasonal infertility of the sows. This seasonal effect is expressed in temperate climates, as a decrease in farrowing rate and prolificacy during the summer and early autumn ,7,8. The®, a commercial semen supplement containing oxytocin, lecilerin, and caffeine (Pat. no. WO2018/1002/14), which increases fertility and litter size following AI in sows [® remains to be tested in other breeds, particularly Iberian sows. Iberian pigs are reared in Spain for their cultural and gastronomic value and ability to adapt to harsh environments [We have shown that semen doses supplemented with oxytocin, cloprostenol, and buserelin increased litter size and fertility and helped to ameliorate seasonal infertility ,10. This in sows . Nonetheronments ,13. Howeronments  for a co® could be used to improve fertility in Iberian sows, particularly when bred in the hotter months typically associated with reduced reproductive performance.Therefore, the objective of this study was to investigate whether SuinfortThe University of León Animal Care Committee reviewed and approved the protocol and procedures. The study was performed with Iberian sows located in a farrow-to-finish farm near Valverde de Mérida  for two consecutive years (June 2016 to August 2018). Sows between 8 and 98 months old were housed in individual gestation stalls and had boar contact for 5–10 min/d for up to 15 d to facilitate detection of the first post-weaning estrus. They remained in the stalls after AI and until confirmed pregnant 35 d after mating. Pregnant sows were housed in pens with 38 sows/pen until they were moved to individual farrowing crates from 1 week before farrowing until weaning at 28 d. Coolers and artificial lighting kept farrowing room temperatures between 18 and 34 °C throughout the year with a minimum daily photoperiod of 12 h.9 sperm in 80 mL extender. All semen doses were used within 48 h of collection. At estrus detection, sows were assigned by parity to receive the seminal additive Suinfort containing 2 IU oxytocin, 5 µg GnRH analog (lecirelin), and 2 mM caffeine  or serve as non-supplemented controls . In total, 4338 AI were carried out in 1430 sows (interquartile range: 2–5 AI per sow), with an average parity of 4.9 ± 2.8 (mean ± SD). The SF was included in the seminal dose 15 min before the first insemination only, as we have previously shown no additional effect of seminal supplement inclusion in the second insemination [Semen was collected weekly from 68 Duroc boars (12–30 months of age) using routine protocols. The sperm quality was checked at the stud farm following standard protocols, with all having acceptable levels of motility (≥80%) and morphology . Sows were artificially inseminated  at estrus detection and 24 h intervals while exhibiting estrus with 3 × 10p ≤ 0.05.Data analysis was performed in the R statistical environment v.4.0.4 . Fertilip = 0.05). Parity had a significant effect , with no interaction with the treatment. We observed a decrease in fertility for CON during spring and summer compared with winter ; there was some recovery by autumn (p = 0.012 with summer), although fertility remained significantly lower than in winter (p = 0.022). SF reduced this trend and was only the summer that was significantly lower than winter (p = 0.005). Comparing the groups within each season, fertility was significantly higher in the SF group during both summer (p = 0.025) and autumn (p = 0.004).Farrowing rates following AI  were affp < 0.001) and winter . Stillborn piglets . Mummified piglets (p = 0.036) and winter (p ≤ 0.001). Parity significantly affected total born and live-born with p < 0.001 ; stillborn and mummified with p = 0.010 (1-farrowing group with lower numbers). The For sows that farrowed, total born, live-born, stillborn, and mummified piglets  did not  piglets c decreas piglets d decreas–pituitary–ovarian axis would mirror those for conventional genotypes.The present data demonstrate the effect of season on Iberian sow fertility, with both farrowing rates and litter sizes being lower in summer and autumn. The effects of season on the fertility of typical commercial sows have been reviewed, and it was suggested that both temperature and photoperiod are involved in the seasonal effects on sow fertility . One lin® on sow fertility although, in this study, the effect was only significant in the summer and autumn months, possibly due to an amelioration of the effect of heat stress on Iberian sows. Seasonal infertility represents a problem for many pig breeding systems, with heat stress impacting sow ovarian activity and oocyte developmental competence [Consistent with our previous work , in the mpetence ,18. Thesmpetence ,13,19. Hmpetence ,21. Iber® in the Iberian sows are likely attributable to the constituent components. The oxytocin may decrease the retrograde flow of sperm while promoting uterine contractions, helping to establish an optimal oviduct sperm reservoir [®, since caffeine stimulates the motility of both fresh and cryopreserved sperm [® may have promoted a better synchronization of ovulation, explaining the improvements noticed in summer and autumn compared with CON pigs [®-treated Iberian sows.The effects of Suinforteservoir ,23,24. Aed sperm ,26. Whiled sperm . TherefoCON pigs . Taken t® improved fertility throughout the year of Iberian sows inseminated with Duroc semen. Further studies should investigate whether Suinfort® can be used to improve fertility in Iberian sows inseminated with Iberian semen and other non-commercial autochthonous breeds.The present data demonstrate that seminal supplementation with Suinfort


The primary cilium is a solitary, microtubule-based membrane protrusion extending from the surface of quiescent cells that senses the cellular environment and triggers specific cellular responses. The functions of primary cilia require not only numerous different components but also their regulated interplay. The cilium performs highly dynamic processes, such as cell cycle-dependent assembly and disassembly as well as delivery, modification, and removal of signaling components to perceive and process external signals. On a molecular level, these processes often rely on a stringent control of key modulatory proteins, of which the activity, localization, and stability are regulated by post-translational modifications (PTMs). While an increasing number of PTMs on ciliary components are being revealed, our knowledge on the identity of the modifying enzymes and their modulation is still limited. Here, we highlight recent findings on cilia-specific phosphorylation and ubiquitylation events. Shedding new light onto the molecular mechanisms that regulate the sensitive equilibrium required to maintain and remodel primary cilia functions, we discuss their implications for cilia biogenesis, protein trafficking, and cilia signaling processes. Primary cilia are dynamic cellular signaling compartments of the plasma membrane  composedPost-translational modification (PTM) is a fundamental principle in molecular biology referring to the modulation of protein properties by covalent attachment of small molecules. PTMs are catalyzed by various antagonistic enzymatic activities that modify target proteins at specific locations . For insThe dynamic nature of PTMs is critical for most cellular processes and is extensively studied in protein trafficking and cell signaling . The cenConceptually, primary cilia are believed to function as cell type-specific micro-compartments with diverse compositions including receptors to receive, mediators to process, and effectors to transmit signals to the rest of the cell . DespiteOne hallmark ciliary signaling pathway that highlights the dynamics in PTMs is Hedgehog signaling in vertebrates . An elegUpon Hedgehog ligand binding, PTCH1 exits the primary cilium and SMO is activated and retained in cilia, whereas GPR161 is removed . As the One central element in Hedgehog signaling is the dynamic re-localization of the components involved. Similar to other cellular protein trafficking mechanisms, PTMs control the localization of Hedgehog signaling proteins, which is particularly well-studied for GPR161 . GPR161’Drosophila  . TTBK2 lon assay . Additioon assay . UBR5 haon assay , where ion assay . It willon assay , are plaChlamydomonas, allowing efficient cilia disassembly (In contrast to cilia formation, we are just beginning to understand the molecular details of how cilia are dismantled to allow cell cycle re-entry . NEK2, aassembly . In turnassembly . While tChlamydomonas, a ubiquitin conjugation system has been identified more than a decade ago (Chlamydomonas model has only detected an increase in ubiquitylation of α-tubulin and ubiquitin itself (In cilia of cade ago , yet then itself . The stun itself . Intrigun itself .As we are gathering increasing evidence for the existence of a ciliary ubiquitylation machinery involved in protein trafficking, signaling, disassembly, and potentially protein quality control, its identity remains elusive. Similarly, antagonistic cilia-specific DUBs as well as protein phosphatases that counterbalance known kinases await their identification. Powerful unbiased genetic and proteomic screening technologies have been applied to primary cilia  and promEM wrote the first draft of the manuscript. TS conceptualized and prepared the figures. DM wrote sections of the manuscript. All authors contributed to the conception of the article, manuscript revision, and read and approved the submitted version.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.


Uric acid serum levels correlated with negative sub-score , general sub-score , and total PANSS score  after 1 month of therapy. We have established a statistically significant positive correlation between serum concentrations of UA and IL-6 in exacerbation  and with IL-17 after treatment and in the stabilization of psychosis , suggesting potential cascades in different phases of schizophrenia that potentiate inflammation.Uric acid (UA) has been shown to have neuroprotective or neurotoxic properties, in relation to specific tissues and diseases that have been studied. Previous studies provided contradictory results on the role of UA in schizophrenia as a neurodegenerative disorder. The aim of this brief report was an additional analysis of UA sera levels in different phases of schizophrenia. Here, 86 patients with first-episode psychosis (FEP) vs. 45 patients with schizophrenia in relapse (SC in relapse) vs. 35 healthy control subjects (HC) were studied before and 1 month after antipsychotic therapy. Further, we aimed to explore the possible correlation of UA with scores presenting clinical features and with serum concentrations of the proinflammatory cytokines interleukin (IL)-6 and IL-17. When comparing the data between all three groups, we did not find significant differences in UA levels, either before or after the applied therapy. Also, comparing sera concentrations of UA in every single group, the analysis did not reveal statistically significant differences between FEP patients, but statistically, a significant difference was found in SC in relapse before and after treatment (334.71 ± 116.84 vs. 289.37 ± 109.15 μmol/L,  Uric acid (UA) is the final oxidation product of the adenine- and guanine-based purine catabolic pathway and is considered as a risk factor of numerous pathological conditions . HomeostIncreased levels of UA in the initial phase may reflect an attempt to counteract oxidative stress, suggesting its neuroprotective role , whereasThe relationship between altered UA levels and schizophrenia remains unclear, and studies have yielded conflicting results. A significant decrease in plasma UA levels was observed in the first episode of schizophrenia , and a mThis study represents an additional analysis of the database from our previous cytokine studies in different phases of schizophrenia , 20. BriA somatic examination was performed, and vital signs were measured on admission. Patients' blood samples were collected at least 8 h before the administration of antipsychotic therapy during the current psychotic episode. The control group consisted of HC subjects matched to the patients by gender and age with patients and recruited at a blood donation in the Service supply of blood and blood products, of Clinical Center Kragujevac. Serum UA levels were determined using a hematology analyzer, ABX MICROS 60-OT (UK). The reference values for UA were in the following range: 154–428 μmol/L. Serum cytokine levels were measured using conventional enzyme-linked immunosorbent assay (ELISA) kits . Immunological measurements were performed at the Center for Molecular Medicine and Stem Cell Research, Faculty of Medical Sciences, University of Kragujevac. The acute effects of treatment on UA serum levels were assessed after 4 weeks, since the first 2–4 weeks of antipsychotic treatment are crucial for a substantial reduction of positive symptoms .p-value of 0.05 was considered to be statistically significant.Statistical analyses were performed using SPSS 20.0 software . Numerical values are reported as mean, standard deviation (SD), and standard error (SE). The normal distribution of the data was tested using Shapiro–Wilk and the Kolmogorov–Smirnov test. A Paired Sample test and the Wilcoxon test were used to compare the clinical parameters in the same group before and after therapy. We used the Mann–Whitney test for non-parametric variables to assess mean differences between two groups of patients. A Kruskal–Wallis test was used to assess differences in serum UA levels between three groups . We examined the correlations between serum UA and cytokines, as well as the relationship between serum UA levels and PANSS scores, positive, negative, and general psychopathology sub-scores using Pearson's or Spearman's correlation. Multiple regression analysis was used to assess the significant demographic and clinical predictors of serum UA levels and PANSS scores. A From our previous study database , 20, onlp = 0.663) and after applied therapy , we did not detect statistically significant differences in UA values before therapy. When comparing serum UA levels in each group, the analysis did not reveal statistically significant differences between FEP patients , but statistically, a significant difference was found in SC in relapse, before and after treatment . We also analyzed serum UA levels between FEP and SC in the relapse group before treatment , and after treatment , and no statistically significant differences were observed , general sub-score , and total PANSS score  after 1 month of therapy in both patient groups, while a correlation between serum UA levels and positive sub-score was not significant   and with IL-17 after treatment and in the stabilization of psychosis  (We found a significant correlation between UA serum levels with negative sub-score (= 0.149) . In addi = 0.34) .F(3/108) = 8.027, p < 0.001, R2 = 0.187, Adjusted R = 0.163] was a significant predictor of serum UA levels, with female gender implicating an increase in UA. Uric acid serum levels  and IL-17 serum levels after treatment in all psychotic patients  are significant predictors for PANSS score (presented in Multiple regression analysis indicated that sex [In the new tendency to publish negative results, we must emphasize that we did not find statistically significant differences in serum UA levels between patients with FEP, SC in relapse, and HC subjects, neither before nor after the applied therapy. We pointed out that patients with schizophrenia had higher UA serum levels in exacerbation than in the stable phase of this psychosis. There is a significant correlation between UA serum levels and negative, general sub-score, and the total PANSS score in the stabilization phase. A statistically significant positive correlation was observed between serum concentrations of UA and IL-6 in exacerbation, and with IL-17 after treatment and in the stabilization of psychosis. Our findings suggest that in a stable phase of the illness, serum levels of UA and IL-17 are significant predictors for PANSS scores.Firstly, we would like to discuss phase specificities of oxidative and inflammatory stress in schizophrenia. Plasma UA levels were significantly lower in patients with FEP, but not in patients with bipolar or depressive disorder . In contAntipsychotics could increase the levels of UA . In a stIn addition, the immune response seems to be modulated through UA impact. We found a statistically significant positive correlation between serum concentrations of UA and IL-6 in exacerbation and with IL-17 after treatment and in the stabilization of psychosis. Interleukin-17 is a pro-inflammatory cytokine produced mainly by Th17 cells and Type3 innate lymphoid cells when stimulated by cytokines such as IL-6 or IL-1, stimulating the production of cytokines and chemokines, and facilitating inflammation . An incrYao et al. discussed the previously established links between UA and intelligence and reported the data from their research on the association between low levels of UA and greater impairment in sensory processing tasks . In a moWhen examining whether these cascades might be of clinical benefit in patients with Parkinson's disease, lower UA levels seem to imply a higher risk of transition to mild cognitive impairment . In contThis study reflects the naturalistic sampling in real clinical practice, in a specific timeframe. The diagnosis could be made only after a longer prodromal phase of the disease, sometimes to avoid stigmatization of these patients and due to inadequate family support. The limitation of this study is also that the data do not allow extrapolation in the chronic patients and required further research of a much longer duration of the illness. In addition, the fact that the decrease in FEP patients was not statistically significant could be a by-product of the higher variance in the FEP group, which also requires a larger sample size.Our results indicate a possible detrimental effect of elevated serum UA levels in certain phases of schizophrenia. No significant differences in UA levels were observed in the initial phase of the illness, but it has been shown by others that repeated acute relapses might be followed by increased UA levels in patients with a longer duration of illness. Uric acid serum levels could be associated with IL-6 elevation in exacerbation, once more showing IL-6 as a crossroads in the pathophysiology of schizophrenia. These results could be explained by the fact that a different type of immune response is activated in schizophrenia than in other neurodegenerative diseases. For example, multiple sclerosis predominates type 1 immune response, while in schizophrenia type 2 immune response overweights and type-17 immune response could be related to cognitive decline. It could be considered that the type of immune response influences the further role of UA and whether it exhibits pro/antioxidant or pro/anti-inflammatory effects. Also, it is important to consider the lower serum levels of UA after therapy in patients with chronic schizophrenia, and that this potential further elevation of UA may contribute to residual symptomatology and poorer therapeutical response. Uric acid can be considered as a marker of the disease course in the later disease cascades.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The study involving human participants, was reviewed and approved by the Ethics Committee of the Clinical Centre Kragujevac and the Special Hospital for Psychiatric Diseases “Dr. Laza Lazarevic”, Belgrade, and was conducted in compliance with the ethical principles. Informed consent was obtained from all patients before any study procedures began.MB and the team of trained psychiatrists have selected the patients, performed the psychological assessment, and sampling. MB has conceptualized the manuscript together with KV. KV and MB did the statistical analysis. KV designed figures and tables. MB and SJ wrote about the psychiatric aspects of this topic. KV and NM wrote about the role of UA. IJ and NA did the cytokine measurements and wrote about immunological aspects. MB prepared an integral version of the manuscript. All authors contributed equally to the content of this manuscript and approved the final version of the manuscript.This work was supported by the Ministry of Science and Technological Development of the Republic of Serbia, No. 175069, and by the Faculty of Medical Sciences, University of Kragujevac, Nos. JP 03/16 and JP 12/09.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Triple-negative breast cancer (TNBC) is not as prevalent as hormone receptor or HER2-positive breast cancers and all receptor tests come back negative. More importantly, the heterogeneity and complexity of the TNBC on the molecular and clinical levels have limited the successful development of novel therapeutic strategies and led to intrinsic or developed resistance to chemotherapies and new therapeutic agents. Studies have demonstrated deregulation of Wnt/β-catenin signaling in tumorigenesis which plays decisive roles at the low survival rate of patients and facilitates resistance to currently existing therapies. This review summarizes mechanisms of Wnt/β-catenin signaling for resistance development in TNBC, the complex interaction between Wnt/β-catenin signaling, and the transactivated receptor tyrosine kinase (RTK) signaling pathways, lymphocytic infiltration, epithelial-mesenchymal transition (EMT), and induction of metastasis. Such associations and how these pathways interact in the development and progression of cancer have led to the careful analysis and development of new and effective combination therapies without generating significant toxicity and resistance. Triple-negative breast cancer (TNBC) cells are identified by the lack of receptor targeted therapies including human epidermal receptor 2 (HER2), estrogen receptor (ER) and progesterone receptor (PR), and are major cause of lack of better therapies, consisting about 15–20% of recently diagnosed breast cancer [Based on tumors’ characteristics including size, morbidity, and the number of involved lymph nodes, chemotherapeutic agents have been the primary systemic options for TNBC patients . HoweverWnt ligands activate distinct intracellular signaling, which can be categorized into β-catenin-dependent and β-catenin-independent pathways Fig. . AccordiAlthough many reports have suggested that TNBCs tend to respond well to chemotherapeutic drugs, patients may frequently develop a chemorefractory and poor prognosis. Interestingly, recent findings lend support to the possibility that targeting Wnt/β-catenin signaling in combination with chemo- and targeted- therapies in patients with TNBC exhibits highly synergistic results and prevent Wnt signaling function in chemoresistant cancer cells. According to the in vitro studies of Xu et al. , β-catenBRCA1/2 mutation to excrete Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) actively out of the cells [FZD1 gene expression, by siRNA, reduced cytoplasmic/nuclear β-catenin level and P-gp overexpression.Several mechanisms can lead to the development of chemoresistance, among which ATP-binding cassette (ABC) transporters are the most thoroughly validated. Three transporters have been extensively identified in TNBC comprising (i) multidrug-resistant protein-1 and -8 (MRP1 and 8), (ii) breast cancer resistance protein (ABCG2), and (iii) the P-glycoprotein (MDR1) pump which pumps a wide array of chemotherapeutics out of the cancer cells. Previous studies have demonstrated increased nuclear and cytoplasmic β-catenin accumulation induced ABCB1 (MDR1 or P-gp) in TNBC involving he cells . Therefohe cells , frizzleMany studies have shown that cancer stem cells (CSCs) are more resistant to conventional therapies in TNBC, which cause survival and relapse of cancer. Moreover, it has been shown that hypoxia inducing factor 1-α (HIF-1α) enhances Wnt/β-catenin signaling in the hypoxic microenvironment and promotes β-catenin stabilization and upregulation of CSCs transcription genes . HoweverThese observations were supported by the elevated activation of the Wnt signaling pathway following stimulation of drug inactivation/detoxification, deregulation of DNA damage repair pathways, cell cycling, and apoptosis. Consequently, as Wnt signaling becomes aberrantly activated in cancer cells, these signaling mechanisms become perturbed resulting in the development of resistance to selected TNBC’s therapeutic agents. Therefore, there is an urgent clinical need to better understand diverse mechanisms of resistance to multiple conventional and targeted cancer therapies through elevated Wnt signaling and to introduce novel combinatorial therapies extending TNBC disease-free intervals.So far, multiple crosstalks between Wnt signaling and other intracellular signaling pathways have been identified in TNBC, which can cause resistance to therapies. The gene-expression profiles of TNBC cells have shown an association between EGFR and Wnt signaling in TNBC patients treated in the adjuvant setting. Recent findings on the mechanisms of resistance in TNBC cells, which constitutively overexpress EGFR, demonstrate that silencing Wnt/β-catenin signaling contributes to the reduction of the levels of β-catenin, RAS, and EGFR and put forward the possibility of combinatorial treatment for TNBC patients . Given tThe interaction between EGFR signaling activation and the Wnt/β-catenin pathway involves series of kinase signaling cascades for the interruption of adherent junctions. Most important examples include crosstalk between Wnt/β-catenin and EGF/RAS/ERK signaling pathways which involve accelerating β-catenin nuclear translocation through GSKGSA-3β inhibition , 20. ConWnt signaling confers EGFR-dependent activation of the PI3K/Akt/mTOR pathway and nucleus localization of β-catenin and EGFR target genes. Akt can activate β-catenin nuclear translocation and reduce GSK-3β activities, which results in increased transcription of proliferative genes, and tumor cell invasion . In addiRecent evidence published by Solzak et al. has indicated that mono-agent therapies (using PI3K inhibitors) are commonly subject to resistant development in the majority of TNBCs. As such, combination therapy against both PI3K and Wnt pathways using buparlisib (panPI3K) and Wnt974 (Wnt-pathway), demonstrates significant in vitro and in vivo synergy against TNBC cell lines and xenografts . MentionIn addition, the vast majority of studies have focused on the Wnt/ROR signaling and its influence on tumor cell function starting with two receptor tyrosine kinase-like orphan receptors (RORs), ROR1 and ROR2 with the activation of non-canonical Wnt signaling responses triggered by binding of Wnt5A ligand , 27. RecPerformed studies on TNBC cells have shown the biological importance of tumor-infiltrating lymphocytes (TILs) and the balance between cytotoxic and regulatory pathways in the tumor microenvironment. It has previously shown that the presence of TILs in the tumor site, in early-stage TNBC correlated with significantly improved clinical outcomes and disease-free and overall survival rates in patients . AberranThe interplay between positive and negative immunoregulatory signals is carried on to ensure that the adaptive immune system is capable of defending the host while maintaining self-tolerance and preventing autoimmunity and one important aspect is the PD-1/PD-L1 axis. PD-1 and PD-L1 are immune check-point ligand and receptor which restrict T cell effector functions within tissues . TNBC ceIn addition, increasing studies indicated up-regulation of CSC-related Wnt signaling pathway and tumor-intrinsic activation of β-catenin signaling in PD-L1High TNBCs , 38. Thehigh/CD24low stem-like cells in a set of mammary cancer cell lines [Cancer cell dissemination and metastasis are well-established as the worst trait of cancer progression and are correlated with therapeutic resistance. Loss of E-cadherin and accumulation of nuclear β-catenin induce CD44ll lines . Considell lines , 41. As ll lines  revealedll lines . In addill lines , 46. TakTNBC is extensively studied over the last few decades on account of its unique clinical pathologies, such as larger tumor size, nodal involvement, high distant recurrence rates, and molecular features. Currently, targeted therapy in combination with chemotherapy is approved to treat locally advanced or metastatic TNBCs. Therefore, screening more reliable biomarkers is imperative, and understanding the signaling pathways that regulate biological behaviors may facilitate the establishment of effective therapeutic strategies. Wnt/β-catenin signaling’s crosstalk with TNBC-associated tumorigenic mechanisms results in amplification of their underlying signaling cascades and development of resistance to different therapeutic agents. Consequently, establishing new promising therapeutic regimens with specific targeting abilities of its upstream and downstream signaling seems to be an attractive challenge for overcoming resistance and prevention of cancer progression. Studies in preclinical and clinical trials have shown that the concurrent inhibition of Wnt signaling together with administration of cancer chemo- and targeted- therapies develop synergistic effects in the treatment of TNBCs. Despite several signs of progress in understanding the contributing role of Wnt signaling in the development of TNBC and possible mechanisms underlying the development of resistance to anti-cancer agents, still, many questions remain unsolved which must be answered in near future to precisely design safe combinational therapeutic strategies with high potency and low toxicity.


G protein-coupled receptors (GPCRs) are the largest family of cell-surface proteins. They are characterized by seven transmembrane domains; a cytoplasmic C-terminus, and an extracellular N-terminal domain. GPCRs respond to a wide variety of signaling molecules and signal through the trimeric G-protein complex. GPCRs are expressed essentially on all cells, facilitating cellular responses to external stimuli and are involved in nearly every biological process . There aThis research topic comprises of research papers and review articles on diverse GPCRs and their regulators. The collection of articles highlights the role of GPCRs in physiology, pathophysiology, and explores the possibility of exploiting their therapeutic potential. Drosophila. They demonstrate that AdoR signaling represents an important pathway in response to stress conditions such as cytotoxicity, oxidative damage, and thermal stress in Drosophila. The paper provides important insights into the molecular mechanism of Ado regulation of stress response that could help understand how Ado signaling affects disease pathogenesis.An interesting paper by In a comprehensive review , criticain vivo efficacy. The data from this study add to the earlier experimental data on the ability of KK-92A to reduce nicotine self-administration and cue-induced reinstatement of nicotine seeking in rats and therefore, broadening the anti-addictive profile of KK-92A.It is widely documented that GPCR signaling involves cross-regulation of many pathways including cross-talks between different GPCRs as well as with other signaling pathways. Besides the acute signaling, GPCR, in direct or through crosstalk, also regulate the development of addictive diseases. In this area , in theiThe review by Finally , describIn conclusion, the research topic contains a fascinating collection of original research papers and review articles encompassing cell biology, physiology, and molecular pharmacology of G-protein coupled receptors. We hope that the data and information conveyed through this research topic will be beneficial to the scientific community in general and researchers in this exciting research area, in particular. We believe the research ideas presented will push more studies to further understand the physiological significance of GPCRs and unearth additional therapeutic possibilities.


The relative expression of ELF3, chondrocyte degeneration-related molecules, matrix metalloproteinase, and inflammatory molecules were detected in chondrocytes stimulated with interleukin (IL)-1β or co-incubated with SMSC-212-5p-Exos or SMSCs-derived exosomes (SMSC-Exos). IL-1β induced up-regulation of ELF3, down-regulation of degeneration molecules , up-regulation of matrix metalloproteinase , and up-regulation of inflammatory molecules  could be inhibited by SMSC-212-5p-Exos or SMSC-Exos administration. When compared with the SMSC-Exos, SMSC-212-5p-Exos showed more treatment benefits. All of these indicate that SMSC-212-5p-Exos could suppress chondrocyte degeneration and inflammation by targeting ELF3, which can be considered as a disease-modifying strategy.Excessive chondrocyte degeneration and inflammation are the pathological features of osteoarthritis (OA), and altered miR-212-5p may contribute to meniscus and cartilage degeneration. Whether exosomes derived from miR-212-5p overexpressed synovial mesenchymal stem cells (SMSC-212-5p-Exos) could be utilized to treat degenerative chondrocytes is investigated in this study. Down-regulated miR-212-5p and up-regulated E74 Like ETS Transcription Factor 3 (ELF3) expression were detected in OA synovial tissues, which showed a negative correlation ( Osteoarthritis (OA) is a disease that comprises progressive cartilage degenerative and synovial membrane inflammation, having an increased frequency of osteophyte formation and subchondral bone sclerosis in the aging population . No curaExosomes are small size (30–100 nm), single-membrane organelles released by various cells to shunt loading bioactive molecules to target cells . ResearcMicroRNAs (miRNAs) can post-transcriptionally regulate OA-associated genes in chondrocytes . ExosomaBased on the criteria of the American College of Rheumatology, thirty OA patients  underwent total knee arthroplasty were enrolled. Meanwhile, the synovial tissues were collected from OA patients and 20 donors with accidental deaths (excluding those with OA-related diseases) or normal knee-joint synovium who were subjected to lower limb amputation after acute trauma or open reduction and internal fixation after fracturing of the tibial plateau . The study was approved by the Xijing Hospital, and informed written consent was derived from each participant or close relatives.® easyCyte™ flow cytometer . At passage 3, SMSCs were switched to osteogenic differentiation medium  for 2 weeks or StemPro Adipogenesis Differentiation Kit (Gibco) for 4 weeks. The miR-212-5p mimic or mimic-negative control (NC) (Sigma-Aldrich) were transfected with Lipofectamine® 3,000 (Thermo Fisher) at the concentration of 100 nM according to the manufacturer’s instructions. The following sequences were used: miR-212-5p mimic , mimic-negative control miRNA .Synovial membrane specimens were digested with 0.2% type I collagenase (Thermo Fisher) at 37°C overnight, which were further collected by centrifugation and seeded in a high-glucose DMEM medium  supplemented with 10% FBS for 4 days to allow cell attachment. The medium was refreshed every 3 days, and at day 14, SMSCs were obtained. After blocking with human BD Fc Block™, SMSCs were stained with the following antibodies (Becton Dickinson): anti-CD34, anti-CD44, anti-CD45, anti-Sca-1, and anti-CD105 antibodies to confirm the phenotype using GuavaConditioned medium of SMSCs or miR-212-5p overexpressing SMSCs was filtered with 0.22 μM filters (Merck-Millipore) and centrifuged  to concentrate the volume into approximately 200 μL, which was further ultracentrifuged  to obtain the exosomal particles. A transmission electron microscope (TEM) was utilized to observe the morphology of exosomes, and the size and distribution of exosomes were measured using dynamic light scattering (DLS) analysis.7) were seeded into the 6-well plates 1 day before treatment. When cell confluence reached 80%, 2 µg exosomes (EXO miR-NC and EXO-miR-212-5p) were introduced into the chondrocytes culture medium supplemented with 10 ng/ml IL-1β. Chondrocytes treated with PBS were regarded as the blank controls. After 48 h of treatment, cells were collected for subsequent use.Primary chondrocytes were detached from cartilages dissected from the subchondral bone with 0.25 mg/ml collagenase P and 4 mg/ml protease, and the isolated chondrocytes  was applied to detect the amplification. The primers were listed as following: ELF3, forward 5′- CATGACCTACGAGAAGCTGAGC-3′, reverse 5′- GACTCTGGAGAACCTCTTCCTC-3’; miR-212-5p, forward 5′- CAGTCTCCAGTCACGG-3′, reverse 5′- GAACATGTCTGCGTATCTC-3’; Collagen II (COL2A1), forward 5′- CCTGGCAAAGATGGTGAGACAG-3′, reverse 5′- CCTGGTTTTCCACCTTCACCTG-3’; Aggrecan, forward 5′-CAGGCTATGAGCAGTGTGATGC-3′, reverse 5′- GCTGCTGTCTTTGTCACCCACA-3’; Sox9, forward 5′- AGGAAGCTCGCGGACCAGTAC-3′, reverse 5′- GGTGGTCCTTCTTGTGCTGCAC-3’; MMP-1, forward 5′- ATGAAGCAGCCCAGATGTGGAG -3′, 5′- TGGTCCACATCTGCTCTTGGCA -3’; MMP-3, forward 5′- CACTCACAGACCTGACTCGGTT-3′, reverse 5′- AAGCAGGATCACAGTTGGCTGG-3’; MMP-13, forward 5′- CCTTGATGCCATTACCAGTCTCC-3′, reverse 5′- AAACAGCTCCGCATCAACCTGC-3’; IL-6, forward 5′ AGACAGCCACTCACCTCTTCAG-3′, reverse 5′- TTCTGCCAGTGCCTCTTTGCTG-3; COX-2, forward 5′- CGGTGAAACTCTGGCTAGACAG-3′, reverse 5′- GCAAACCGTAGATGCTCAGGGA-3’; iNOS, forward 5′- GCTCTACACCTCCAATGTGACC-3′, reverse 5′- CTGCCGAGATTTGAGCCTCATG-3’; GAPDH, forward 5′- CTGTGCCGTTGAATTTGCCG-3′, forward 5′- CGGGTTCCTATAAATACGGACTG-3’.TRIzol (Invitrogen) was utilized to extract total RNA from chondrocytes or SMSCs. Total Exosome RNA & Protein Isolation Kit  was used to extract RNA and protein from exosomes for further analysis. TaqMan™ Advanced miRNA cDNA Synthesis Kit (Thermofisher) was utilized to reverse-transcript miRNA into cDNA, and RNU6B was utilized as an internal reference. TransscriptThe QuikChange Lightning Site-Directed Mutagenesis kit  was utilized to mutate the binding site of ELF3 with miR-212-5p. 3′-UTR fragment of ELF3 mRNA (mutant or wide type) was subcloned into pGL3 luciferase vector , which was further co-transfected with miR-212-5p mimic or mimic-NC into HEK-293T cells for 36 h with Lipofectamine 3,000. Subsequently, the luciferase activity was assayed with Luciferase Assay System (Promega).The cellular lysates were quantified using the BCA protein concentration kit , and 20 µg lysates were separated with 10% SDS-PAGE electrophoresis and transferred to PVDF membranes, which were further blocked in Tris-buffered saline with Tween 20 (TBST) containing 0.1% Tween 20 and 5% skimmed milk powder and followed by incubation with the primary antibodies  specific for CD63, CD9, Alix, ELF3, and GAPDH, and then incubated in peroxidase-conjugated secondary antibody (Sigma-Aldrich) at room temperature for 1 h. The dilution used for the primary antibodies against CD63, CD9, Alix, and ELF3 was 1:1,000, the dilution used for the primary antibody against GAPDH was 1:2000, and the dilution used for the second antibody was 1:2000. The signal was developed with an ECL system , and the relative intensity was normalized with GAPDH expression with NIH-Image J1.51p 22.According to the manufacturer’s instructions, the concentrations of IL-6, MCP-1, MMP-3, MMP-13, and TNF-α were detected with commercial ELISA kits . Separated stock standard solutions and samples were measured with a SpectraMax M5 microplate reader at a wavelength of 450 nm.p-value < 0.05 was considered statistically significant.Statistical analysis were performed with Graph-Pad Prism 6.0. Data were presented as means ± SD. The Mann-Whitney test was performed to determine the statistical significance between any two groups, and Dunn’s multiple comparisons test was used to compare multiple groups. p < 0.001), and up-regulated ELF3 mRNA expression (p < 0.001) and increased protein expression of IL-1β (p < 0.001) were also observed. The expression correlation analysis indicated that both the correlation between ELF3 and miR-212-5p  and the correlation between IL-1β and miR-212-5p  were significantly negatively correlated. As expected, the correlation between ELF3 and IL-1β was positively correlated .The relative miR-212-5p expression in OA synovial tissues was down-regulated compared with healthy control (Spindle-like SMSCs were observed at passage 3 (P3) , which cTargetScan  and miRBUp-regulated miR-212-5p expression could be detected in SMSC-212-5p-Exos , indicatThe relative mRNA expression of Collagen II , AggrecaThe relative content of IL-6 , MCP1 F, and TNFIn addition to inflammation, dysregulation between the synthesis and degradation of extracellular matrix mainly mediated by type-II collagen, aggrecan, and matrix metalloproteinases contributes to the articular cartilage degradation and loss process . In thismiR-132/212 clusters are maintained on down-regulation in mesenchymal stem cells at four different stages of transforming growth factor-β3-induced chondrogenesis differentiation . On the As transcription factors belonging to the ETS family, ELF3 participates in autoimmune and tumor neogenesis . The prein vivo models should be constructed to demonstrate the therapeutic benefit of SMSC-212-5p-Exos. Whether other molecules, except miR-212-5p, could contribute to the treatment advantage in SMSC-212-5p-Exos compared with SMSC-Exos need further detailed analysis.ELF3 is identified to direct target miR-212 to suppress nasopharyngeal carcinoma cells proliferation . In thisin vitro model is utilized in the study, and the OA in vivo model should be constructed to demonstrate the treatment benefit of SMSC-212-5p-Exos. All in all, this study indicates that SMSC-212-5p-Exos could target ELF3-mediated OA pathology in chondrocytes, which can be considered as a future treatment option.Accumulating investigations have indicated that exosomes will pave a new therapeutic paradigm for osteoarthritis treatment. Our study testifies that SMSC-212-5p-Exos could alleviate IL-1β induced chondrocytes degradation, degradation, and inflammation process involved in OA. It is worth noting that only


Peritoneal dialysis (PD) is one of the most important kidney replacement therapies for patients with end‐stage kidney disease (ESKD). PD technique failure can lead to an escalated cost and increased infectious and cardiovascular risk, up and including to death. The accumulation of uric acid (UA) was associated with adverse outcomes in ESKD patients. However, the relationship between serum UA and technique failure is little explored.Here, a total of 266 continuous ambulatory peritoneal dialysis (CAPD) patients  were enrolled and followed up for 31.7 months. Serum UA levels were examined at baseline and each visit. Subjects were divided into three groups according to their baseline serum UA concentrations. Multivariable Cox regression models were used to estimate the hazard ratios (HRs) and 95% confidence intervals (CIs) of PD technique failure.p for trend was 0.043. Adjusted HRs of all-cause technique failure, transferring to HD and mortality with each 1 mg/dL increase in serum UA were 1.20 , 1.22 , and 1.25 , respectively.The level of serum UA increased gradually as time prolonged. During the follow-up period, 77 (28.9%) patients occurred PD technique failure, of which 56 (21.1%) transferred to hemodialysis (HD) and 21 (7.9%) died. Compared to the lowest UA tertile, after adjusting for potential confounders, HRs of technique failure in tertile 2 and tertile 3 were 1.82 (95% CI: 0.95–3.49) and 2.03 (95% CI: 1.05–3.92), respectively, and Higher serum UA level predicted higher risk of technique failure in CAPD patients. Peritoneal dialysis (PD) is a cost-effective kidney replacement therapy in end-stage kidney disease (ESKD) patients . CompareTechnique failure of PD caused by peritonitis decreased peritoneal solute transfer rate, ultrafiltration failure, circulatory overload, heart failure, or cardiovascular events, finally leading to transferring to HD or even death ,6. UndesUric acid (UA), the end product of purine metabolism in humans, is excreted two-thirds by the kidneys . Due to The present longitudinal research was conducted to investigate the long-term trends of serum UA, and explore the relationship between serum UA and PD technique failure in continuous ambulatory peritoneal dialysis (CAPD) patients.The PD patients were recruited from a single-center cohort study based on continuous ambulatory peritoneal dialysis patients in central China. During 2013–2020, 266 CAPD patients in Tongji Hospital were recruited with the following select criteria: (1) receiving CAPD treatment longer than 3 months; (2) aged more than 18 years; (3) no diabetic nephropathy; (4) no severe malnutrition ; (5) no diagnosis of gout or receiving urate-lowering drugs; (6) no pregnancy; (7) able to provide consent.www.chictr.org.cn, registration ID: ChiCTR1900024905). Informed consent was obtained from participants before the study in accordance with the declaration of Helsinki.The study was approved by the Institutional Review Board of Tongji Hosipital, Huazhong University of Science and Technology (TJ-IRB20201010), and registered at clinicaltrials.gov , and female . The longitudinal trend of circulatory UA and excretion of UA were investigated over time.2). Hypertension was recorded if the patient took antihypertensive drugs or had two separate blood pressure measurements ≥140/90 mmHg. Medicine uses of antihypertension drugs, UA lowering drugs, and erythropoietin were recorded according to the prescriptions and adherence of the patient. The biochemical variables including blood urea nitrogen (BUN), serum creatinine, total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), blood glucose, hypersensitive C-reaction protein (hsCRP), albumin, prealbumin, hemoglobin, potassium, calcium, phosphorus, magnesium, sodium, were measured with auto-biochemistry analyzer machine at the central laboratory of Tongji Hospital.Demographic and comorbidities data, including age, sex, weight, height, systolic blood pressure, diastolic blood pressure, primary kidney disease, edema, hypertension, were collected from the electronic medical records at the initiation of PD. Body mass index (BMI) was calculated as weight divided by height squared  was used to evaluate dialysis adequacy and were calculated from a 24-h collection of dialysate and urine with the use of standard methods, dialysis adequacy was diagnosed as Kt/V ≥ 1.7 . Weekly Work flow of this cohort study is shown in N) and percentage (%). One-way ANOVA or Kruskal–Wallis test were used to compare differences among three groups for approximately normally distributed continuous variables or skewed distribution variables, respectively. Differences in categorical variables were tested using Chi-square test. A Kaplan–Meier analysis was used to evaluate the change in survival among the tertiles of serum UA, and curves were compared using the log-rank test. Cox proportional hazard regression models were used to investigate the unadjusted and multivariable adjusted associations between serum UA level and PD technique failure. To maximize statistical power to examine the relationship between UA level and PD technique failure, continuous variable analyses were conducted with hazard ratios presented by per 1 mg/dL UA increase. Additionally, Cox regression models were repeated with UA as a continuous variable in stratified analyses to exam for statistical interaction between UA and several confounding factors through multiplicative interaction terms, p < 0.1 was considered significant [The generalized additive models (GAM) were used to curve fit the trend of UA levels over time in CAPD patients. Participants were stratified into three groups by sex-specific tertiles of baseline serum UA level. Summary statistics are presented as mean ± standard deviation for approximately normally distributed continuous variables, median with interquartile range (IQR) for continuous variables of skewed distribution. Categorical variables were presented as number  were enrolled in this prospective cohort study. At baseline, the mean age of the patients was 41.92 years, and the median PD duration was 6.45 (IQR: 4.19–13.95) months. The baseline biochemical parameters were compared among three tertile groups. As shown in The longitudinal trend of circulatory UA and excretion of UA are shown in Supplementary Table S1), lower serum albumin, higher serum UA, and lower RKF were associated with technique failure in CAPD patients, while the hazard ratios (HRs) and 95% confidence intervals were 0.91 (0.86–0.96), 1.18 (1.03–1.36), and 0.48 (0.25–0.93), respectively. The Kaplan–Meier survival curves for PD technique failure in patients with different serum UA levels are illustrated in p = 0.022).The median follow-up period was 31.7 months (IQR: 12.1–55.4 months). During the follow-up period, 56 CAPD patients were transferred to HD therapy, 21 patients occurred death, of which 10 patients (47.6%) died of cardiovascular events. Overall, the incidence rate of PD technique failure was 28.9%. According to the results of the univariate and multivariate Cox regression models  of serum UA concentration was associated with a higher risk of transferring to HD therapy, and p value for trend was 0.043. The risk of mortality was higher in tertile 2 compared to tertile 1 group in multivariable Cox regression model 3, but without significance.p = 0.019), 1.22 , and 1.25 , respectively.Furthermore, serum UA was examined as a continuous variable. As shown in p for interaction was 0.013). All other factors included in the analysis showed nonsignificant interactions.The effect of high serum UA on PD technique failure was further performed by stratified analysis. The entire cohort was divided to subgroups by gender or the median value of age, BMI, PD duration, albumin, RKF, and Kt/V. As shown in In the present study, we observed the level of serum UA increased gradually as the peritoneal dialysis time prolonged while the total excretion of UA tended to be stable. Furthermore, higher serum UA level at baseline predicted a higher risk for PD technique failure.Since a large part of UA is excreted through kidneys, it is conceivable that decreased kidney function is accompanied by increased serum UA. PD therapy is used to eliminate wastes and toxins accumulated in ESKD patients. However, our results indicated that the serum UA of CAPD patients elevated gradually over time. The decreased urine UA possibly attributed to the declined RKF as the dialysis time prolonged. In total, only 200–250 mg/day UA was eliminated in CAPD patients, even though the dialysate excretion of UA increased over time. The excretion of UA in CAPD patients was far less than 500–750 mg/day through urine in healthy person or more in hyperuricemia patients with normal kidney function ,24. The Patients with higher UA level were associated with higher phosphorus, triglyceride and lower HDL-C in the present study. It was similar to Xiang’s report that they found higher serum UA level was independently associated with higher serum albumin, higher BMI, higher phosphorus concentrations, which reflected nutritional status . AdditioAdditionally, here in this cohort study, patients with diabetic nephropathy were excluded, the results suggested that baseline elevated serum UA level were associated with a higher risk of technique failure in non-diabetic PD patients, which was similar to Xia’s findings that higher serum UA level predicted higher risk of all-cause mortality in non-diabetic PD patients, but no significant association was found in female diabetic PD patients . It implThe underlying mechanisms connecting elevated serum UA level with higher risk of PD technique failure are still far from being well understood. Technique failure can occur for a number of reasons, including peritonitis, inadequate dialysis, ultrafiltration failure, circulatory overload, heart failure, or cardiovascular events. Peritonitis is the leading cause of technique failure and accounts for nearly half of the failures ,39. UricThe strengths of our study are as follows: (1) the prospective cohort study provided longitudinal changes in UA level and a gradually rising trend in serum UA was observed in continuous PD patients; (2) our results remained robust after several stratification analysis and adjustments for multiple potential confounders including demographic characteristics, laboratory data, nutritional status, comorbidities, and medications; (3) patients with complicated comorbidities like diabetes and diabetic nephropathy were excluded to avoid the potentially confounding factors associated with poor outcomes. There are also several limitations in this study: (1) it was conducted in a single center and had a relatively small sample size, the number of death events were relatively limited, and the statistical power was insufficient to draw a final conclusion about the association between serum UA and mortality in these patients; (2) only CAPD patients without diabetes were recruited in this study, caution should be taken when generalizing the conclusion to other populations; (3) as an observational study, the associations do not prove causality, the underlying mechanisms linking elevated serum UA and technique failure needed further clarification.In conclusion, higher serum UA concentration was associated with higher risk of technique failure in CAPD patients. It is advisable to integrate serum UA and other clinical characteristics to adjust therapeutic program in advance to prevent worse outcomes. Further studies are needed to explore whether UA-lowering treatment improve technique survival in CAPD patients, and to clarify the underlying mechanisms.Click here for additional data file.