Adaptive interactions in large populations often require honest signals of group membership to structure interactions. However, limitations to a simple mapping of groups onto stylistic and ethnosomatic variation suggest that new ways of measurement are needed to describe the work that objects do to facilitate social coordination. Means to measure the benefits to coordinating on specific objects, here called signaling value, would transition inquiry from general statement that signals play a role, to which signals play what roles in what contexts. This study introduces a method to measure the signaling value of specific objects using classification tasks. After mathematically showing how social coordination leads to greater associations in object classification, a statistical approach is derived to estimate the signaling value of objects from a triad classification task. The approach is then applied to a study of culturally salient motifs in the Pacific Island nation of Tonga and a comparison group in the US. The statistical estimates suggest a large role for social coordination for the full set of motifs, although there is a substantial range of signaling values among motifs. In light of the estimates, the cultural history of individual motifs is discussed as well as the future of this approach. Media summary: Whether we realize it or not, all of us use dress, language, body markings or other outward signs to communicate our ethnic, religious or other group affiliations. If we can decipher the role of specific signals, then we can understand more deeply how human societies organize themselves to coordinate, cooperate and maintain aspects of culture. However because individuals have multiple affiliations that change over time, it is difficult to rank the importance of specific signals. This article addresses this problem with a new approach. By measuring how individuals classify a set of candidate signals, it shows how to estimate the value of each signal. This method is applied to a study of motifs in the Kingdom of Tonga.One of the solutions to the question of large-scale human adaptability includes the structuring of human expression to promote social coordination Goffman, . ObjectsIn large populations social coordination is a formidable problem given the segmented and nested structure of group identities. Multiple ethnolinguistic, political and wealth classes are present with individuals navigating the social milieu to gain benefits with in-group members Cohen,  and infoet al., et al., Famously it was argued that stylistic variation may effectively signal the presence of group boundaries Barth, . HoweverOne major obstacle is that ethnographic studies challenge any simple assigning of objects to groups. In a number of studies there is mixed evidence for clear mapping of stylistic variation on ethnolinguistic groups Hodder, . A vigneOne solution to deciphering the relevance of candidate signals lies in methods describing the construction of categories. Often used to measure a population's response to social, cultural and environmental conditions, object classification tasks are an ethnographic method that describe how populations organize what they observe Weller, . The proTo see how this might work, let us imagine a population of individuals that play coordination games on three objects, {1, 2, 3} – the smallest possible number of objects in a classification scheme. Each individual associates two objects together, either {1,2}, {2,3} or {1,3}, and interacts with others in the population, gaining benefits depending on how many objects match their partner's. Two individuals both with {1,2} gain more benefits than two individuals having {1,2} and {1,3}, given that the former has two objects in common and the latter only one. Further let us also say that some objects lead to greater coordination payoffs than others, if the coordination payoff to object 2 is greater than that to object 3, such that two individuals coordinating on {1,2} have greater payoffs than when they coordinate on {1,3}. Assuming that differential reproduction of the object pairs is an increasing function of these payoffs, then we can solve for conditions when two objects are associated more together than alternatives.δi is the benefit acquired from two individuals coordinating on object i, hereafter called the signaling value, and xij is the frequency of two objects i and j associated together, then the condition for the pair {1,2} to increase in frequency is:Mathematically, if This expression says that the signaling value to object 3 must be less than a linear combination of the signaling values to objects 1 and 2 scaled by the frequency and/or variance of relevant pairs . In other words, two objects may be classified together if at least one has a higher signaling value and high enough frequency in the population. This relationship is explored in detail in δi) for each object and the extent of social influence on the whole set. The statistical methods below develop this approach, which is then applied to calculating the signaling value of six culturally salient motifs among the Tongan ethnolinguistic group.The link between signaling values and object pairing as demonstrated by this model provides an opportunity. If object classification reflects socialization through coordination benefits, we can reverse-engineer object classification data to measure the signaling value  even if the person knows nothing of the objects. The consequence is that individuals will group objects by features that have no connection to social coordination, e.g. stylistic similarities.A = {1, 2, …, NA} a common approach is to take a subset B of three objects from A (B⊆ A) and ask a respondent to pair two objects as most similar by choosing the most dissimilar object , by the fraction of pairs made in the triad classification task across Nu individuals in this population. This follows from the multinomial probability function, where the fraction of the time a pair is chosen empirically is equal to the maximum likelihood estimate of its probability of being chosen in a multinomial probability model. Thus,Inherent classification may be estimated using data collected in populations previously not exposed to the objects. We can estimate the probability of two objects, e.g. {1, 2} out of three {1, 2, 3} associated together, Nu individuals indexed by k in the unexposed population, yk = 1 if the pair {1, 2} is grouped together, zero otherwise. This can be computed for any pair within a triad.where across all f. xij owing to payoffs in a coordination game. As in the theoretical model above , parameK1, K2) and Pr for any pair {i, j} to give its frequency in the next generation. By simulating the frequency of x12, x13 and x23 to equilibrium, we explore the long-run expression of each pair of objects as a function of signaling value (δi).From x12) as a predicted probability that a pair of objects is grouped together from a set of three, constructing f(coordination) in equation   Helu, .While model estimates suggest that the set as a whole is under strong social influence, the signaling values are relative to others in the set and thus inferences are limited by the set of objects classified. It is key, therefore, to identify salient objects and understand their contextual limitations. As the theoretical and statistical models introduced here assume that the signaling values are stable attributes of the population, the analysis should be applied with a comparative approach that captures the spatiotemporal dynamics of signals.Thus while the motifs studied here have undoubted ethnographic importance among Pacific cultures Ewins, , future u is underestimated. This may be solved by choosing an object set that reduces redundancy by eliminating motifs that may be playing the same social role.Because it is possible that stylistic similarities between motifs are caused by their coordinated signaling value, then this paper might underestimate the social coordination value of the motif set. This follows from the choices of the unexposed reference population, which if they classify based on stylistic similarity, will cause the model in  to attriThere are probably better data collection protocols to measure signaling value. For example, one may devise various prompts, from the more conservative and ambiguous ‘which object is most different?’ used in this study to the more directed ‘which object doesn't belong here?’ The latter may elicit stronger social categorization of objects than what is found in this study. Also, other variations of the classification task and object clustering may be used as an ethnographic method, e.g. pile sorts. The clustering approach may then allow social value to be estimated for an arbitrary cluster of objects, rather than just single objects. A new statistical specification will be needed to model the clustering possibilities and signaling values.The ethos of the future work outlined above may follow the sizable literature and data collection effort on semantic representations (Nelson, McEvoy, & Schreiber, et al., Once the value of objects can be measured then general discussions of group signaling can be transformed into an object-centered discussion that relates object signaling values to theoretical expectations of signaling dynamics, and/or align with ethnographic significance. The analysis of Tongan motifs demonstrates that these possibilities are in reach, relating signaling values to history and contemporary forces of cultural change. Using this method more complex predictions may be addressed relating nested or evolving group boundaries Wimmer, , demogra


Post-stroke depression (PSD) is a common psychiatric complication of mental disorders after stroke. Acupuncture for PSD is effective and has few adverse effects. As a classical complementary and alternative therapy, acupuncture is often used in combination with antidepressants for PSD. However, there is a wide variety of acupuncture therapies, and the efficacy of different acupuncture varies. In this study, a network meta-analysis (NMA) was used to assess the clinical efficacy of different acupuncture combined with antidepressants for the treatment of PSD.A comprehensive search of PubMed, The Cochrane Library, EMbase, Web of Science, CNKI, CBM, VIP, and Wan-Fang databases for published randomized controlled trials of acupuncture combined with antidepressants for the treatment of PSD was conducted. The time frame for the literature search was from the date of database creation to April 30, 2022. The Cochrane risk of bias tool for randomized trials (RoB 2.0) was used to evaluate the bias risk of the included studies. Data analysis was performed by STATA 14.0 software.A total of 38 literatures with 2,898 patients involving nine acupuncture therapies were included. NMA results were as follows: moxibustion plus antidepressants had the best efficacy in terms of improving total effective rate. Conventional acupuncture plus antidepressants was the most effective in improving HAMD scores. In terms of improving SDS scores, acupressure plus antidepressants was the most effective. In terms of improving NIHSS scores, moxibustion plus antidepressants showed the best results.A comparison of the efficacy indicators of the nine different acupuncture therapies combined showed that moxibustion plus antidepressants, conventional acupuncture plus antidepressants and acupressure plus antidepressants were superior in the treatment of PSD. Based on the shortcomings of the existing studies, this conclusion needs to be validated by additional high-quality randomized controlled trials. The At present, the main drugs for clinical treatment of PSD are monoamine oxidase inhibitors, tricyclic antidepressants, selective 5-hydroxytryptamine reuptake inhibitors and new antidepressants developed in recent years . The useAcupuncture is often used in combination with antidepressants for PSD. Studies have confirmed that acupuncture combined with antidepressants is superior to antidepressants alone . Huang’s2.NMA was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines .2.1.2.1.1.A randomized controlled trial (RCTs) of acupuncture combined with antidepressants versus antidepressants for PSD will be included. The publish language is limited to English and Chinese.2.1.2.Included patients were diagnosed with PSD and were not limited in gender, race, comorbidity, or duration of disease.2.1.3.The control group used antidepressants alone. The experimental group used acupuncture combined with antidepressants, and acupuncture-related therapies included conventional acupuncture, moxibustion, electroacupuncture, warm acupuncture, ear acupuncture, fire acupuncture, etc.2.1.4.The primary outcome indicators were total effective rate and Hamilton Depression Scale (HAMD) score. If the HAMD score decreased by more than 75% compared with that before treatment, it showed that it was a cure; if the HAMD score decreased by 50–75% compared with that before treatment, it showed an obvious effect; if the HAMD score decreased by 25–49% compared with that before treatment, it showed an effective result; if the HAMD score decreased by less than 25% compared with that before treatment, it showed that it was ineffective. Total effective rate = cure rate + obvious effective rate + effective rate .Secondary outcome indicators were National Institutes of Health Stroke Scale (NIHSS) and Depression Self-Rating Scale (SDS) scores. The included literature had clear and relevant evaluation criteria.2.2.(1) Duplicate published literature.(2) No clear diagnostic criteria and no efficacy criteria.(3) Literature with incorrect data or inaccessible data.2.3.A comprehensive search of PubMed, The Cochrane Library, EMbase, Web of Science, CNKI, CBM, VIP, and Wan-Fang databases for published randomized controlled trials of acupuncture combined with antidepressants for the treatment of PSD was conducted. The time frame for the literature search was from the date of each database creation to April 30, 2022. MeSH terms were paired with free terms, using the appropriate Boolean logical operator connections for the search, and appropriate adjustments were made according to the different databases to include all retrieved literature. For Pubmed, for example, the specific search strategy is described in 2.4.The literature was screened, extracted and cross-checked by 2 investigators independently. In case of disagreement, the decision was made by the corresponding author. For literature screening, the title of the text was read first, and after excluding apparently irrelevant literature, the abstract and full text were further read to determine inclusion. Enter the data needed by this research by excel. Data was extracted from: (i) basic information of the included studies, including study title, authors, year and month of publication, sample size, and diagnostic criteria; (ii) interventions, including types and duration of antidepressants and acupuncture; (iii) characteristics of study subjects; (iv) evaluation entries and results of risk of bias evaluation; and (v) outcome indicators, including total effective rate, HAMD score, NIHSS score, and SDS score.2.5.Risk of bias evaluation of the included RCTs by 2 evaluators according to the Cochrane risk of bias tool for randomized trials (RoB 2.0) . Items e2.6.χ2-test evaluated the heterogeneity between the results of the included studies, and I2 quantified the heterogeneity. When p ≥ 0.1 and I2 < 50%, homogeneity was indicated and a fixed-effects model was selected for analysis; conversely, a random-effects model was used. Subgroup analysis according to treatment duration (>6 weeks and ≤ 6 weeks). Sensitivity analysis to test the stability of meta-analysis results. If no less than 10 papers were included, small sample effects or publication bias were detected by comparison-adjusted funnel plots. Since this study is an indirect comparison of acupuncture combined with antidepressants versus antidepressants for PSD, no consistency test is required. Statistical analysis was performed using Stata 14.0 software and its network, mvmeta package, etc., to draw a net relationship diagram, funnel diagram, etc. for comparison between interventions. The effects of the interventions were ranked by surface under the cumulative ranking curve (SUCRA); larger SUCRA values were ranked higher, indicating that the intervention was more likely to be the most successful one.Dichotomous variables were expressed using relative risk ratio (RR) and its 95% confidence interval (95% CI). Continuous variables were expressed using standardized mean difference (SMD) and its 95% CI. The 3.3.1.After initial review of 3,446 relevant literature, 38 literatures with 2,898 patients were finally included after stratum-by-stratum screening. The literature screening process and results are shown in 3.2.A total of 2,898 patients with 38 literatures , 31–60 i3.3.10.52% of studies had low risk of bias, 13.16% of studies had high risk of bias and the remaining 76.32% had unclear risk of bias . The mai3.4.3.4.1.p < 0.05). The results of descriptive analysis showed that the total effective rate of moxibustion plus antidepressants was statistically significant compared to antidepressants (p < 0.05). While the total effective rate of auricular pressure plus antidepressants compared with antidepressants, the difference was not statistically significant (p > 0.05). The results of pairwise meta-analysis are shown in The results of the meta-analysis showed that electroacupuncture plus antidepressants, abdominal acupuncture plus antidepressants, auricular electroacupuncture pl-us antidepressants, conventional acupuncture plus antidepressants and auricular pressure plus antidepressants were statistically significant differences in total effective rate compared with antidepressants (3.4.2.p < 0.05). While theHAMD scores of auricular electroacupuncture plus antidepressants and electroac-upuncture plus antidepressants were not statistically significant when compared with antidepressants (p > 0.05). The results of descriptive analysis showed that HAMD scores of wrist-ankle acupuncture plus antidepressants and auricular pressure plus antidepressants were statistically significant when compared with antidepressants (p < 0.05). While the HAMD score of eye acupuncture plus antidepressants was not statistically significant when compared with antidepressants (p > 0.05). The results of pairwise meta-analysis are shown in The results of the meta-analysis showed that electroacupuncture plus antidepressants, abdominal acupuncture plus antidepressants, conventional acupuncture plusantidepressants and moxibustion plus antidepressants were statistically significant differences in HAMD scores compared with antidepressants (3.4.3.p < 0.05). The results of pairwise meta-analysis are shown in The results of descriptive analysis showed that wrist-ankle acupuncture plus antidepressants, acupressure plus antidepressants, auricular pressure plus antidepressants and electroacupuncture plus antidepressants were statistically significant differences in SDS scores compared to antidepressants (3.4.4.p < 0.05). In contrast, there was no statistically significant difference between the HAMD scores of auricular electroacupuncture plusantidepressants and electroacupuncture plus antidepressants compared with antidepressants (p > 0.05). The results of the descriptive analysis showed a statistically significant difference in NIHSS scores for moxibustion plus antidepressants compared to antidepressants (p < 0.05). The results of pairwise meta-analysis are shown in The results of the meta-analysis showed that abdominal acupuncture plus antidepressants, conventional acupuncture plus antidepressants and electroacupuncture plus antidepressants were statistically significant differences in NIHSS scores compared with antidepressants (3.5.p < 0.05). The results of the subgroup analysis are shown in To further explore the effect of treatment duration on outcomes, a subgroup analysis was conducted in this study. Due to the small amount of literature involved in some acupuncture treatments, it was impossible to perform subgroup analyses for all outcome indicators. Therefore, only the total efficiency and HAMD scores involved in the conventional acupuncture plus antidepressants group were analyzed in subgroups. Subgroup analysis showed that conventional acupuncture plus antidepressants was statistically different in terms of increasing total effective rate and improving HAMD scores for both treatment duration >6 weeks and treatment duration ≤6 weeks  standardize acupuncture point selection treatment standards and standardized operation guidance  in future research; (2) conduct long-term follow-up to judge the long-term curative effect and recurrence of the disease; (3) carry out RCTs of direct comparison between different acupuncture therapies.The original contributions presented in the study are included in the article/CL and MS: conceptualization, software, writing–original draft, and writing—review and editing. SC, SL, and YM: data collection. MS: funding acquisition, resources, and supervision. All authors contributed to the article and approved the submitted version.This work was supported by the Scientific Research Project of Sichuan Provincial Health Commission (20PJ261).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.


Functional neurological disorder (FND) is characterized by neurological symptoms that lack congruence with traditional neurological diagnoses. Historically viewed through a Freudian psychoanalytic lens, FND has been conceptualized as a purely psychogenic disorder. However, the contemporary biopsychosocial perspective on FND emphasizes contributions of cognitive and neural circuit dysfunction and the disabling and involuntary nature of the illness. In Saudi Arabia, evidence suggests the prevalence of FND is significant. However, clinical programs and research focused on FND have been lacking. Studies from the region indicate that practitioners may have outdated views of FND. To address this, this narrative review provides an updated perspective on FND that is relevant to Saudi Arabia and the region. It delves into the evolving perception of FND, its underlying pathophysiology, risk factors, clinical presentations, and recent diagnostic and management advances. Unique features of FND in Saudi Arabia may include a significant role for family disputes as a risk factor, prevalent supernatural perceptions of FND, high prevalence of somatization, and cognitive dysfunction, and a potential favorable prognosis. The article concludes by providing the following recommendations related to FND in Saudi Arabia and the region: i) building educational programs to update clinicians about contemporary biopsychosocial perspectives on FND; ii) emphasizing a positive diagnostic approach based on clinical findings in FND; iii) instituting multidisciplinary programs to care for FND patients; iv) supporting systematic research efforts to explore culture-specific FND risk factors, patient outcome measures, and attitudes toward the disorder; v) developing national FND clinical practice guidelines; and vi) launching awareness campaigns to reduce FND stigma. Functional neurological disorder (FND) is a neuropsychiatric disorder characterized by neurological symptoms that are incongruent with neurological diagnoses . Also knFND has garnered considerable attention from clinicians and researchers over the past decade, leading to advancements in the understanding and management of the disorder . This waThis narrative review aims to provide an updated practical perspective on FND that is relevant to Saudi Arabia and the Arab Middle East. The aim is to describe how the perception of FND has evolved, its pathophysiologic underpinnings, risk factors, clinical profile, and recent advances in how it is diagnosed and managed. This paper examines the landscape of FND research in Saudi Arabia and concludes by providing recommendations regarding how FND programs and research may be developed in the region.History of FND: from the antiquities to the Diagnostic and Statistical Manual of Mental Disorders (DSM)Medical and cultural interpretations of paroxysmal neurological symptoms have evolved over time . In prehThe concept of FND has subsequently evolved with the emergence and evolution of the DSM . In the Contemporary perceptions of FND: neural circuit dysfunction and the biopsychosocial perspectiveAlthough the pathophysiology of FND is far from being adequately understood, evidence from a large body of neuroscience research suggests that the deficits seen in FND involve dysfunction in neural networks. For excellent reviews on this, the reader is encouraged to read the reviews by Drane et al. and Milano et al. ,19. FiguAn integral component of FND’s neural circuit pathology is the disturbance circuitry of affective regulation. There is evidence of increased activation of the amygdala and periaqueductal gray and dysregulated prefrontal top-down control of these limbic structures. This may explain increased and sustained emotional reactivity in FND. Furthermore, limbic and motor control circuits appear to have increased functional connectivity, explaining how involuntary movements or weakness may occur. Another notable dysfunctional neural circuit in FND is that of self-agency, a person’s sense of ownership of their own movements and perceptions. After a movement is performed, feedback signals travel from sensorimotor areas to multimodal sensory areas  to generate the sense of self-agency of that movement. Studies have shown right temporoparietal dysfunction in patients with motor FND, leading to a lack of ownership of performed movements . Other dThe nature of neural network dysfunction in each patient is thought to dictate the emergent FND symptomatology, thus creating the FND core symptom subtypes. FND symptoms may therefore be neurologically localizable to the dysfunctional circuits, even if lesions are not seen in routine neuroimaging or clinical investigations ,19.Clinical features: the era of positive diagnosisFND may present acutely, chronically, or in a relapsing manner. An inciting physical or psychological traumatic event may take place but is not required for the diagnosis . There iFunctional weakness often presents as hemiparesis or quadriparesis but may affect one limb or both legs. PNES presents with seizure-like events, characterized by altered awareness and focal or generalized limb shaking without associated epileptiform EEG changes. Functional abnormal movements may present with assimilating tremors, gait unsteadiness, slowing of movement, sudden jerks, and dystonia-like posturing. Functional speech abnormalities include hoarseness slurred speech, stuttering, or speaking with a foreign language or accent. Functional sensory loss may affect any body part and does not align with known sensory pathways. Hemianesthesia that abruptly stops at the midline (midline splitting) is typical. Functional visual disturbances present as intermittent blurred vision, double vision, or eye movement abnormalities. Functional cognitive dysfunction may present with memory issues, word-finding difficulties, and mental ‘fogging’. Severe memory loss and fugue states may be seen. These symptoms are distinguished from cognitive symptoms accompanying mood disorders and anxiety by the loss of insight in the former and its presence in the latter ,17.In addition to the main functional symptoms, patients may display other characteristic symptoms or signs. Patients may exhibit La belle indifference, a lack of concern incongruous with the symptom, and severity and disability. Unexplained pain syndromes are commonly seen and should raise the possibility of FND in patients with motor FND or functional movement disorders ,2. In adStudies suggest that the symptomatology of FND in Saudi Arabia and the Middle East may have some unique features, although studies are insufficient to draw firm conclusions. Although core FND symptoms likely share more similarities than differences across cultures, region-specific features, and culture-bound syndromes have been described ,21,22. TAlthough the diagnosis of FND requires careful consideration of neurological diagnoses, FND should not merely be thought of as a diagnosis of exclusion. Positive clinical findings consistent with FND should be sought and confirmed. Table Diagnostic criteria for FND syndromes have been proposed and are helpful in guiding the diagnostic process ,27. PNESManagement of FND: the multidisciplinary approachThe initial and crucial step in managing FND is the proper and accurate presentation of the diagnosis to the patient, followed by counseling and education. Proper counseling not only establishes the therapeutic alliance with the patient but may also facilitate acceptance of the diagnosis and directly lead to short-term improvements in FND symptoms. The first essential element when presenting the diagnosis is to avoid accusing patients of faking their symptoms or blaming them for their inability to control these symptoms. The best approach is to explain FND symptoms from a biopsychosocial perspective, highlighting how the brain’s normal functioning may be disrupted by psychosocial stressors, maladaptive coping mechanisms, neurodevelopmental factors, and trauma. The approach of incorporating neurobiological aspects of the disorders’ pathophysiology may reduce the likelihood of rejecting the diagnosis and improve their participation in the treatment plan -30.Clinicians should then consider the role of psychotherapy, particularly if patients do not improve upon follow-up after the initial diagnosis presentation. Cognitive-behavioral therapy (CBT) was shown in clinical trials to improve FND outcomes -33. CBT Psychologically minded rehabilitation approaches have proven to be a useful component of multidisciplinary care in FND ,36. PhysOther treatments may be considered in FND. Pharmacotherapy may be helpful in treating comorbid symptoms of FND . AntidepBecause optimal diagnosis and management of FND often involve multiple healthcare providers from different disciplines, it is crucial that these professionals work together as a team. The team may include professionals from neurology, psychiatry, psychology, physical therapy, occupational therapy, speech and language therapy, social work, and others. Multidisciplinary care ensures a seamless patient journey, reduces inconsistent messages from different professionals, and has been shown to be associated with improvement in physical and psychological symptoms, and shorter hospitalization ,15,37.FND in Saudi Arabia: insights and recommendationsAlthough systematic epidemiological studies focused on FND in Saudi Arabia and the region are lacking, FND appears to be prevalent in Saudi Arabia. The author has observed in an unpublished retrospective chart review that about 10% of patients seen at an academic neuropsychiatry clinic in Saudi Arabia had FND, a prevalence equivalent to that seen in other parts of the world . Cross-sThe literature suggests the possibility of unique culture-specific features of FND in Saudi Arabia. First, the risk factor profile may be different or incompletely understood. In our yet unpublished retrospective chart review of FND, family disputes were the most common psychosocial risk factors, and sexual abuse was reported by 15% of patients. Similar findings were reported in an Iranian sample of FND patients ,41. In pThere may also be unique features in the clinical symptomatology of FND in Saudi Arabia but studies are insufficient. In the author’s unpublished retrospective study, cognitive dysfunction was prevalent and may have been associated with negative outcomes upon follow-up. Patients in this sample reported a high prevalence of supernatural explanations of FND symptoms. Reported supernatural themes included possession, sorcery, divine punishment, and others. These beliefs may have clinical implications and may be associated with stigma ,42. SomaAnother intriguing point of interest is how cultural and religious beliefs may influence patient acceptance and response to FND. Based on religious culture, Saudis invariably believe that the soul  can interact with and influence the body. This belief is based on mind-body dualism, the philosophical stance that the mind is distinct from but can interact with the brain and the body. This philosophical stance was shown to be common in Saudi Arabia and may be associated with the stigma of mental and neurological disorders ,43. HoweIn addition, the attitudes of clinicians dealing with FND in Saudi Arabia are worthy of exploration. In unpublished research, the author and colleagues studied the attitudes of a sample of Saudi neurologists and psychiatrists and found that nearly a third of participants endorsed a predominantly psychogenic view of the disorder, with underemphasis on the disabling and involuntary nature of the disorder, and the neural network dysfunction that is now known to be part and parcel of FND. In this sample, this psychogenic perspective on FND was associated with reduced willingness to participate in multidisciplinary FND care. These attitudes may alter patient outcomes -30.Finally, to the authors’ knowledge, there are no formal, multidisciplinary specialized FND services in Saudi Arabia. In addition, there are no clinical practice guidelines that guide clinicians and institutions when it comes to how to deliver FND care. There may be a wide variation in practice patterns among institutions and individual healthcare professionals taking care of FND . In the In conclusion, FND is a prevalent problem worldwide. Although large-scale epidemiologic research is lacking, available several small studies suggest that FND is prevalent in Saudi Arabia. FND has not received a proportionate degree of focus from clinicians and healthcare systems in Saudi Arabia. FND management hinges upon proper diagnosis presentation and effective multidisciplinary care delivered by a compassionate healthcare team. These aspects may need to be emphasized when planning and providing FND care in Saudi Arabia. There may be unique features in how Saudi FND patients present and how they may perceive and deal with the disorder.The field of FND has moved rapidly over the course of the past couple of decades. This review article presented vital recommendations and a roadmap to improve FND clinical services and research in Saudi Arabia and the Middle East Arab countries, and many of these recommendations would apply at a global level as well. Examples of sorely needed FND research programs include epidemiological research to study the prevalence and risk factors of the disorder, neuroscience research to elucidate potential biomarkers, and clinical research to validate FND diagnostic tools and outcome measures, as well as research looking to find the best psychotherapeutic and counseling approaches to FND in Saudi Arabia. There is a pressing need for developing, disseminating, and implementing training programs and clinical practice guidelines. Substantial efforts, resources, and leadership are needed to achieve these goals in Saudi Arabia and worldwide.


The use of high-pressure technologies is a hot topic in food science because of the potential for a gentle process in which spoilage and pathogenic microorganisms can be eliminated; these technologies also have effects on the extraction, preservation, and modification of some constituents. Whole grapes or bunches can be processed by High Hydrostatic Pressure (HHP), which causes poration of the skin cell walls and rapid diffusion of the anthocyanins into the pulp and seeds in a short treatment time (2–10 min), improving maceration. Grape juice with colloidal skin particles of less than 500 µm processed by Ultra-High Pressure Homogenization (UHPH) is nano-fragmented with high anthocyanin release. Anthocyanins can be rapidly extracted from skins using HHP and cell fragments using UHPH, releasing them and facilitating their diffusion into the liquid quickly. HHP and UHPH techniques are gentle and protective of sensitive molecules such as phenols, terpenes, and vitamins. Both techniques are non-thermal technologies with mild temperatures and residence times. Moreover, UHPH produces an intense inactivation of oxidative enzymes (PPOs), thus preserving the antioxidant activity of grape juices. Both technologies can be applied to juices or concentrates; in addition, HHP can be applied to grapes or bunches. This review provides detailed information on the main features of these novel techniques, their current status in anthocyanin extraction, and their effects on stability and process sustainability. Non-theHigh Hydrostatic Pressure (HHP) is the application of pressure to food by a pressurizing fluid, usually water, inside a highly resistant steel vessel . InitialHHP, unlike thermal treatments, does not have enough energy to affect covalent bonds; therefore, small molecules with sensory impact are protected, and HHP can be considered a gentle technology. The formation of unexpected molecules has not been observed during HHP processing . FurtherMany studies have shown that using HHP at room temperature preserves nutritional value and has a very low effect on the levels of anthocyanins in fruits and vegetables . The HHPUltra-High Pressure Homogenization (UHPH) is a high-pressure technology in which a fluid is pumped at more than 200 MPa  through a thin, high-resistance steel pipeline and later depressurized via a special valve made of a high-resistance alloy ,20. The Some interesting effects of submicron nanofragmentation are the elimination of microorganisms, inactivation of enzymes , increase in antioxidant activity, and better colloidal stability ,21,22,23High-pressure techniques can be considered gentle, non-thermal technologies with specific features to preserve the nutritional and sensory quality of food products. However, they have several specifications regarding the processing conditions , the requirements of the products to be processed (particle size and liquid/solid), the antimicrobial capacity (pasteurization or sterilization), the inactivation of enzymes, and the effects on molecules with sensory impact . MicrobiUHPH has the advantage of being continuous  with higHHP is a discontinuous or batch process but can be applied to whole grapes, bunches, or grape pomace ,26. The https://www.ypsicon.com/ (accessed on 24 August 2023)). The special high-strength tungsten carbide valves with ceramic or carbon nano coverings can be scaled up to several of these flow ranges. The electric motor of UHPH pumps, which is the most important consumption range, is 11–18 kW for pumps of 60–150 L/h .Industrial scale-up of high-efficiency UHPH systems is now possible with existing UHPH pumps capable of continuous operation at 300 MPa or higher with a pressure imbalance of 1 MPa (<1%). UHPH pumps are available from 60 L/h to 10,000 L/h, covering the range from pilot systems to industrial devices (https://www.hiperbaric.com/en/hpp-technology/ (accessed on 24 August 2023)). There are also semi-continuous bulk HHP systems for liquids able to work at over 4000 L/h ([https://www.hiperbaric.com/en/hpp-technology/equipment/hpp-in-bulk/ (accessed on 24 August 2023)).There are currently several companies , Avure , Uhde , Kobelco  and others ) working000 L/h  . In additituents . We havetituents . The pertituents . The anttituents . A similtituents . The exttituents ,26,34. HHP has shown a protective effect on several anthocyanins with low degradation after treatment  and protThe protective effect on anthocyanins in preserving the antioxidant capacity and the partial inactivation of PPOs make HHP a gentle technique for extracting anthocyanins from fruits or vegetable tissues. The subsequent stability of the extracted anthocyanins under refrigeration favors the stability and valorization of these pigments to be used in the food industry, obtain wines with faster macerations, or even fermentations in the absence of skins, facilitating winemaking.Anthocyanins are essential for the color, antioxidant properties, and sensory quality of berry juices and their fermentative products, such as wine. After juice extraction, anthocyanins can remain entrapped or adsorbed in colloidal particles of the pulp and are also more exposed to the action of PPOs than in the whole berry. Mechanical action and nanofragmentation in the UHPH valve are powerful tools to release the anthocyanins from the colloidal fragments and to avoid the effects of PPOs by inactivating them.In the application of UHPH, the juice is pressurized by pumping with a special pump that works at >200 MPa ; the capillary pipeline section is smaller than 1 mm so that any fluid with a colloid size higher than 500 µm cannot be processed . It woulVitis vinifera L.), yeast cells and large colloidal fragments can be observed  or UHPH for the processing of juices or plant-based beverages ,56,57,58https://www.hiperbaric.com/en/ (accessed on 24 August 2023)). Therefore, treating whole destemmed grape berries or bunches is perfectly possible on an industrial scale [The use of HHP has the advantage of allowing the processing of solids; therefore, it is possible to process grapes, grape pomace, or even bunches. The only limitation is that the size of the product to be treated must be smaller than the diameter and length of the vessel. Large HHP machines with a diameter of 0.38 m can be found on the market today. (al scale , as wellal scale . The extal scale . In addiConcerning anthocyanin oxidation, the main disadvantage of HHP is the variable effect on the inactivation of oxidative enzymes ,27,32,33Lastly, the UHPH technology is environmentally more sustainable than HHP due to the low consumption of water used for processing and cleaning. Sustainability is currently a key parameter in the wine industry ,73,74,75At the same time, the commonly used packaging materials in HHP processing, i.e., polyethylene terephthalate, ethylene–vinyl alcohol copolymer, polyethylene, and polypropylene, could be a negative aspect of this extraction from a sustainability and environmental viewpoint, compared to other emerging extraction technologies such as UHPH , or modeFurther enhancement of high-pressure processing sustainability could be a topic for future studies, which can be achieved by exploring the possibility of using biodegradable packaging materials or developing strategies for re-using and recycling high-pressure processing packaging material. Additionally, there are limited reports on combining high-pressure extraction and other techniques . The posHigh-pressure techniques are a powerful processing tool to enhance extraction in a gentle manner, protecting sensitive molecules such as anthocyanins and maintaining their stability during storage. The inactivation of oxidative enzymes and the absence of oxidation are key process parameters to achieve optimal conditions. As mentioned above, the main advantages of UHPH are its higher antimicrobial efficacy, better control of PPO enzymes, continuous processing, and low consumption of water, detergents, and energy. Conversely, the main disadvantage is the need to process liquid products and the impossibility of processing whole or crushed berries with skins. With regard to HHP, the most interesting processing feature is the possibility to process juice, crushed berries, or whole berries, which is optimal for the extraction of anthocyanins from skins to juice. The main disadvantage is a more variable behavior in the inactivation of enzymes, a less optimal batch process, and a higher consumption of water and energy.2 and obtain food products with clean labels. These HP technologies open up new opportunities to obtain highly sensitive pigments with nutraceutical properties, such as anthocyanins, or to increase their content in juices, smoothies, purees, or derived beverages, even fermented as wines. The appropriate inactivation of oxidative enzymes, the high antimicrobial effect, and the gentle action allow us to reduce chemical additives such as SO


C1041G/+Fbn1) were injected intraperitoneally for several weeks with potassium oxonate (PO), an inhibitor of uricase . Plasma UA and allantoin levels were measured via several techniques, aortic root diameter and cardiac parameters by ultrasonography, aortic wall structure by histopathology, and pNRF2 and 3-NT levels by immunofluorescence. PO induced a significant increase in UA in blood plasma both in WT and MFS mice, reaching a peak at three and four months of age but decaying at six months. Hyperuricaemic MFS mice showed no change in the characteristic aortic aneurysm progression or aortic wall disarray evidenced by large elastic laminae ruptures. There were no changes in cardiac parameters or the redox stress-induced nuclear translocation of pNRF2 in the aortic tunica media. Altogether, the results suggest that hyperuricaemia interferes neither with aortopathy nor cardiopathy in MFS mice.Redox stress is involved in the aortic aneurysm pathogenesis in Marfan syndrome (MFS). We recently reported that allopurinol, a xanthine oxidoreductase inhibitor, blocked aortopathy in a MFS mouse model acting as an antioxidant without altering uric acid (UA) plasma levels. Hyperuricaemia is ambiguously associated with cardiovascular injuries as UA, having antioxidant or pro-oxidant properties depending on the concentration and accumulation site. We aimed to evaluate whether hyperuricaemia causes harm or relief in MFS aortopathy pathogenesis. Two-month-old male wild-type (WT) and MFS mice ( Redox stress (RS) results from the chronic dysfunctional balance between the production and elimination of both reactive oxygen and nitrogen species . RS is largely involved in numerous pathological processes leading to the loss- or gain-of-function of lipids and proteins and changes in gene expression depending on the main subcellular location where it occurs ,2. With FBN1) affecting cardiovascular, ocular, and musculoskeletal systems [MFS is a genetic connective tissue disorder caused by mutations in the fibrillin-1 gene  oxidase 4 (NOX4) aggravates aortic aneurysm progression in both human and mouse MFS aortic samples, and other investigators have subsequently established specific signalling pathways mediating this injury ,26,27. IIn humans and great apes, UA can abnormally accumulate in plasma and some tissues. However, in rodents, UA is quickly catabolised by uricase to allantoin, which is much more water-soluble than UA . In humaUA was found in the aortic walls of human aneurysms and atherosclerotic plaque arteries , and a pTherefore, considering all the above, it might be possible that UA participates, to some extent, in MFS aortopathy, aggravating it by acting as a pro-oxidant and/or promoting ROS production or, just the opposite, mitigating aneurysm progression by acting as an antioxidant. Nevertheless, knowing the broad pathological implication of increased plasma UA levels, we are inclined to think that UA’s effect might be more aggravating than mitigating. Therefore, this study aimed to evaluate the impact of experimentally induced hyperuricaemia in aortic aneurysm progression in a murine model of MFS. Whereas in the observed allopurinol-induced blockade of aortopathy, plasma UA levels remained unaltered and its involvement was initially discarded, this cannot be definitive as, physiologically, UA in rodents is catabolised to allantoin by uricase. Therefore, to study our aim in MFS mice, it was necessary to generate and maintain increased plasma UA levels long enough to coincide with aneurysm formation to evaluate its progression. This transient hyperuricaemic model is feasible utilising uricase inhibitors like potassium oxonate (PO). Our results indicate that, despite the presence of high plasma UA levels, aortic aneurysm progression in MFS mice did not change in any way (aggravating or ameliorating), indicating that UA does not mediate in murine Marfan aortopathy.We first measured plasma levels of UA and allantoin in two-month-old male and female WT and MFS mice , age of p = 0.0016), their respective increases being highly similar and, therefore, not significant between them (interaction genotype/treatment p = 0.9737) (p ≤ 0.0001) but not for the interaction genotype/treatment (p = 0.2966) . Therefore, to study the impact of UA on MFS aortopathy and cardiopathy, we next generated a MFS murine model of hyperuricaemia with regular injections of PO. PO was administered to two-month-old WT and MFS mice for a period of 4, 8, and 16 weeks until three, four, and six months of age, respectively . After f 0.9737) A. Striki 0.2966) B. Some t 0.2966) . Note th 0.2966) . Followi 0.2966) . TherefoJust before blood extraction to measure UA and allantoin levels in each PO-treated and untreated WT and MFS mouse, we analysed the aortic root diameter and cardiac parameters by 2D echocardiography. In three-month-old mice with four weeks of PO treatment, the aortic root diameter in MFS mice exhibited the characteristic aneurysm (WT/PO– vs. MFS/PO–) but showed no differences with PO treatment (WT/PO+ vs. MFS/PO+) . SimilarThe echocardiographic analysis of different cardiac parameters  did not show any significant difference between WT and MFS mice, regardless of the length of PO treatment .We next analysed the structural organisation of the aortic wall in three- and six-month-old mice who received PO treatment for 4 and 16 weeks, respectively . The tunThe nuclear factor erythroid 2-related factor 2 (NRF2) is a key transcription factor that regulates the expression of several antioxidant defence mechanisms. Oxidative stress triggers its phosphorylation (pNRF2), being subsequently translocated to the nucleus to activate the expression response of physiological antioxidant enzymes. Thus, we next evaluated the nuclear presence of pNRF2 in aortic paraffin sections from WT and MFS mice treated with PO for 4 and 16 weeks . Peroxynitrite and 3′-nitrotyrosine (3-NT) products are reliable redox stress biomarkers occurring in the aortic wall of MFS aorta ,25. TherWe also evaluated whether PO treatment might affect systolic blood pressure. This was measured after eight weeks of treatment (four-month-old mice) and presented no alteration .In humans, the role of UA associated with oxidative stress and cardiovascular diseases is under debate. Most evidence comes from epidemiologic studies suggesting that increased serum levels of UA are a risk factor for cardiovascular diseases, knowing that oxidative stress is a relevant pathological mechanism in their pathogenesis and/or progression ,63. AlloIn humans, UA is considered the main antioxidant of the organism and, thus, it has been proposed as a potential treatment for some pathologies that affect the central nervous system ,65. HoweWe have here generated a hyperuricaemic male MFS mouse model following PO administration, and the main results obtained were the following: (i) Despite aortic aneurysm occurring in MFS mice, their plasma UA levels are not significantly different from WT animals; (ii) oxonic acid, supplied as PO, produces a transient but significant elevation in plasma UA levels lasting long enough to evaluate its impact on aortopathy and cardiopathy progression in the MFS mouse model; (iii) PO-induced UA accumulation in blood plasma did not have any impact on either aortic aneurysm progression or the cardiac parameters examined in the MFS mouse model; and (iv) PO-induced hyperuricaemia does not modify the characteristic cellular anti-redox stress response occurring in MFS aortic media revealed by the nuclear translocation of pNRF2 in aortic vascular smooth muscle cells (VSMCs). One striking observation was that plasma allantoin levels did not decrease in parallel with the increase in UA given that PO inhibits uricase activity. We think that this can be explained by the known non-enzymatic transformation of UA into allantoin. Thus, UA can react with anion superoxide, become oxidised and convert into allantoin. Likewise, UA can also be transformed into allantoin under alkaline conditions . The oveDespite evidence supporting a role for RS in human aortic aneurysms, a clear relationship between systemic RS, serum UA levels, total antioxidant capacity, and ascending aortic aneurysms has not been definitively established. Nonetheless, serum UA concentrations and total antioxidant capacity have been reported to be associated with aortic dilatation in humans, suggesting serum UA levels as an indicator of oxidative stress ,73,74,75Uox)-deficient MFS mice would be a more suitable model. We are aware that our study has some limitations. Firstly, the use of rodents to study the impact of UA, whose results are not easily translatable to humans because rodents very rarely accumulate UA. Secondly, we cannot exclude that despite the absence of any effect on aortopathy progression in hyperuricaemic MFS mice, there could be other alterations associated with hyperuricaemia such as kidney damage ,77 or phSodium carboxy methyl cellulose   was prepared with sterile physiological saline as the solvent for potassium oxonate . Potassium oxonate was suspended in CMC-Na. The injected dose was 250 mg/kg and adjusted to the weight of each animal.C1041G/+) were purchased from The Jackson Laboratory . MFS and sex- and age-matched wild-type littermates (WT mice) were maintained in a C57BL/6J genetic background. All mice were housed according to the University of Barcelona’s institutional guidelines . WT and MFS mice were randomly divided into four experimental groups: WT and MFS mice treated with CMC-Na (vehicle), and WT and MFS mice treated with PO. All four groups received the treatment by intraperitoneal injection of the corresponding reagent  every 48 h  with the Spinreact Uric Acid kit (SP41001). We followed the manufacturer’s instructions. g). Ten µL of supernatant was injected into the HPLC system. Separation of allantoin was performed on a Synergy Hydro-RP C-18 reversed-phase column  from Phenomenex . Allantoin elution (at 4 min) was performed with potassium dihydrogen phosphate  and acetonitrile (85:15) with ultraviolet detection (at 235 nm).For the determination of allantoin in blood plasma, an adapted protocol was used, as previously described . BrieflyTwo-dimensional transthoracic echocardiography was performed in all animals under 1.5% inhaled isoflurane. Each animal was scanned 12–24 h before sacrifice. Images were obtained with a 10–13 MHz phased array linear transducer  in a Vivid Q system . Images were recorded and later analysed offline using commercially available software . Proximal aortic segments were assessed in a parasternal long-axis view. The aortic root diameter was measured from inner edge to inner edge in end-diastole at the level of the sinus of Valsalva. Left ventricle (LV) dimensions were assessed in 2D mode in a parasternal long-axis view at both end-diastole (LVDD) and end-systole (LVSD). The interventricular septum and posterior wall thickness at end-cardiac diastole were also measured. All echocardiographic measurements were carried out in a blinded manner by two independent investigators with no knowledge of genotype or treatment.Paraffin-embedded tissue arrays of mice aortae from different experimental sets were cut into 5 µm sections. Elastic fibre ruptures were quantified by counting the number of large fibres breaks in tissue sections stained with Verhoeff–Van Gieson. Breaks larger than 20 µm were defined as evident large discontinuities in the normal circumferential continuity (360°) of each elastic lamina in the aortic media . They we4Cl, 50 mM, pH 7.4) to block free aldehyde groups, followed by a permeabilization step , rinsed three times with PBS and then incubated with 1% BSA in PBS for 2 h at room temperature before overnight incubation in a humidified chamber at 4 °C with the corresponding primary antibody, anti-3-NT  or anti-pNRF2 . On the next day, sections were rinsed with PBS followed by 60 min incubation with the secondary antibody goat anti-rabbit Alexa 647 . Sections were counterstained with DAPI  and images were acquired using an AF6000 widefield fluorescent microscope. For quantitative analysis, four areas from each corresponding tissue section were quantified with ImageJ software (version 1.53). All measurements were carried out in a blinded manner by two independent investigators.For 3-NT and pNRF2 immunofluorescence staining, paraffin-embedded aortic tissue sections (5 μm thick) were deparaffinised and rehydrated prior to unmasking the epitope. To this aim, tissue sections were treated with a retrieval solution  for 30 min in a steamer at 95 °C. Next, sections were incubated for 20 min with ammonium chloride . Mice were positioned on a heating pad and all measurements were carried out in the dark to minimise stress. All animals were habituated to the tail cuff by daily training one week prior to the final measurements. Then, the systolic blood pressure was recorded over the course of three days. For quantitative analysis, the mean value of three measurements per day was used per animal. All measurements were carried out in a blinded manner with no knowledge of genotype or experimental group. p ≤ 0.05 was considered statistically significant. Data analysis was carried out using GraphPad Prism software . Outliers . Firstly, normal distribution and equality of error variance data were verified with Kolmogorov–Smirnov/Shapiro–Wilk tests and Levene’s test, respectively, using the IBM SPSS Statistics Base 22.0 before parametric tests were used. Differences between three or four groups were evaluated using one-way or two-way ANOVA with Tukey’s post hoc test if data were normally distributed and variances were equal, or the Kruskal–Wallis test with Dunn’s post hoc test if data were not normally distributed. A value of Based on our findings, we can conclude that, unlike ROS, UA accumulation seems not to be instrumental in the aortopathy and cardiopathy of a murine model of MFS, at least once the aneurysm is already established and in male mice.


Concomitantly, interleukin (IL)-4 and IL-10 levels significantly increased in people with HUA . In vitro, soluble UA promoted the proliferation and activation of CD4+ T and CD19+ B cells. Thus, HUA is accompanied by elevated peripheral CD4+ T cells and may cause a Th2-dominant immune status.Hyperuricemia (HUA) makes a chronic inflammation status, which affects immune cells. The association between HUA and immune cells, such as monocytes and neutrophils, has been extensively studied. However, studies on HUA and lymphocytes are still limited. We selected 1543 healthy participants and 258 individuals with HUA to analyze the correlation between serum uric acid (SUA) levels and immune cells, and 98 healthy participants and 16 individuals with HUA were used to study the relationship between SUA levels and cytokine levels. Then, we used soluble UA to stimulate peripheral blood mononuclear cells in vitro and examined lymphocyte subset counts and activation by flow cytometry. The results revealed that the number of lymphocytes in the HUA group was significantly increased, particularly CD4 HUA is caused by soluble uric acid (UA) levels in the body exceeding the threshold. As soluble UA levels continue to rise, UA crystals form and deposit in renal tubules, joints, and soft tissues, causing organ and tissue damage and leading to various diseases, the most common of which is gout2. Indeed, gout has been observed in only 36% of patients with HUA. Most patients only exhibit elevated soluble UA levels without deposited UA crystals5. Therefore, HUA deserves more clinical attention.Owing to the improvements in living standards, hyperuricemia (HUA) has become a major public health problem and is currently the second most common metabolic disease after diabetes6. At physiological pH, UA in the body exists mainly in its soluble form7. In the early days of human evolution, soluble UA was broken down by uricase. However, with the loss of uricase, the kidney became the major site for soluble UA excretion; only 10% of soluble UA is excreted in the urine while the remaining 90% is reabsorbed8. This implies that HUA may be essential to the body.Soluble UA that causes HUA is the end product of degradation of purine and purine-containing substances9. The recognition that soluble UA has such key proinflammatory roles in innate immunity raises vital questions of its importance in adaptive immunity. Previous studies have shown that soluble UA increases the expression of immune cell mediator CD40, which connects innate and adaptive immunity by activating dendritic cells, and enhances antigen presentation to T cells11. Moreover, soluble UA increased CD86 expression when co-cultured with primary bone marrow-derived dendritic cells, providing co-stimulatory signals for T cells to initiate the immune response12. Furthermore, depletion of soluble UA suppressed the immune response related to dying cells13. One study on HUA in male individuals described an inverse correlation between serum UA (SUA) levels and natural killer cell counts. Given these findings, we herein explore the relationship between soluble UA, which results in HUA, and lymphocytes (LY).Soluble UA has been found to drive epigenetic reprogramming in innate immune cells, causing a sustained increase in interleukin (IL)-1β gene transcription along with a persistent inflammatory response and increased responsiveness to secondary stimulationHence, we retrospectively analyzed the association of SUA levels with LY and cytokines in healthy participants and individuals with HUA. In addition, we conducted in vitro experiments to further study the effect of soluble UA on LY. Our study not only provides clinical data for determining the relationship between HUA and LY but also provides valuable clues for future research on related mechanisms.P < 0.0001, Fig. P < 0.0001, Fig. P = 0.0303, Fig. We compared the difference in the number of PWBC, LY, NE, and MO and the percentage of LY, NE, and MO between HCs and individuals with HUA. The results indicated that these two groups had similar counts of PWBC in the male population, but the counts of PWBC in the total and female population were significantly higher in the individuals with HUA than in the HCs  vs. 5.86  and 6.36  vs. 5.69 , respectively; both + T, CD8+ T, CD19+ B, and CD16+CD56+ NK cells between the HCs and individuals with HUA. Compared with the HCs, CD4+ T cell counts were significantly increased in females and males with HUA  vs. 821.00  in the total population, P = 0.0019; 694.40  vs. 877.00  in the female population, P = 0.0142; 647.00  vs. 764.00  in the male population, P = 0.0199, Fig. + T and CD16+CD56+ NK cells. Surprisingly, in females with HUA, the number of CD19+ B cells also increased significantly  vs. 295.00 , P = 0.0284, Fig. + T and CD19+ B cell counts in females with HUA tended to be higher than those in males , and no significant changes were found in the levels of IL-2, IL-6, and TNF-α  vs. 2.65  and mean 3.07 ± 0.90 vs. 3.87 ± 1.01, respectively; .05 vs. 2P = 0.0104, P = 0.0108, and P = 0.0484). Soluble UA concentration of 714 µmol/L was associated with an increase in the counts of CD4+ T , CD19+ B , and CD19− NK cells , and soluble UA concentrations of 476 and 238 µmol/L were associated with an increase in the counts of CD4+ T , CD8+ T , and CD19+ B cells  vs. 1.77 , P = 0.0043, respectively). In female patients with fatty liver disease along with HUA aged 41–50 years, LY counts were substantially higher  vs. 1.84 , P = 0.0115, Fig. P = 0.0066, Fig. To explore whether different diseases combination with HUA influences the number of LY, we compared the number of LY in hypertension, heart disease, diabetes, fatty liver disease, and breast cancer with and without HUA. Our findings revealed significantly higher LY counts in patients with fatty liver disease and breast cancer combined with HUA  vs. 2.25  and 1.62  vs. 1.77, respectively; 0 vs. 2.2+ T cell counts and IL-4 and IL-10 levels, implying that HUA may lead to a Th2-dominated immune status.The growing recognition that soluble UA plays a complex role in immune biology has stimulated strong interest in its relationship with immune cells. However, there are few existing analyses of the relationship between soluble UA and LY and even fewer domestic studies; therefore, our study is the first to combine retrospective analyses and in vitro experiments to investigate the effect of soluble UA on LY. We found that elevated soluble UA was mainly accompanied by an increase in CD416. This is in line with our clinical results. CD4+ T cells are divided into Th1 and Th2 cells, of which the latter secrete IL-4, IL-6, and IL-10 to assist humoral immunity17. Therefore, we speculated that the cytokine environment of the HUA population might shift to Th2 cytokine to regulate humoral immunity. Since humoral immunity is mainly mediated by B cells, this is consistent with our in vitro experimental results and further confirms our hypothesis that individuals with HUA may be accompanied by enhanced humoral immunity. However, we need to analyze the relationship between soluble UA and levels of complement and antibodies to further explain our assumption.Studies in patients with HUA have shown that an increase in SUA concentration is related to inflammatory markers such as PWBC, C reactive protein, and circulating innate immune cytokines such as IL-6, IL-1Ra, TNF, or IL-1819. This infers that individuals with HUA are susceptible to allergic and autoimmune diseases. Additionally, it has been reported that the X chromosome harbors genes that can control immune function. Given that females have two X chromosomes, females have stronger immunity and higher levels of physiological antibodies than males20. However, excessive autoantibodies can cause tissue damage, making females more vulnerable to autoimmune diseases. Previous studies showed that females had lower SUA levels compared to males because urate precipitation can be induced by male hormones while it is resisted by female estrogen22. As a result, females may be more sensitive to increased SUA than males. Therefore, females with HUA may develop stronger Th2 immune response than males, accumulating more antibodies with an increased risk of autoimmune disease. This may explain why the number of CD4+ T and CD19+ B cells in females tended to be higher than those in males in our clinical results. Moreover, we found a more significant increase in LY in females aged 41–50 years with fatty liver and breast cancer combined with HUA. This could be because females aged 41–50 years were mostly at menopausal status and had low levels of estrogen, leading to higher levels of SUA, which triggered a stronger immune response.Studies have shown that enhanced Th2-dominated immune response is related to allergic and autoimmune diseases mainly due to the fact that Th2 immune response can promote humoral immunity24. LY counts were increased when HUA was present with fatty liver disease and breast cancer, implying that elevated SUA levels may drive inflammation by promoting LY proliferation. However, we observed no link between SUA and LY in heart disease, hypertension, and diabetes, probably due to the small sample size. In addition, this was a comparative study of immune-related indicators between asymptomatic individuals with HUA and healthy individuals and did not consider whether there were differences between asymptomatic individuals with HUA and those with gout. Therefore, in the future, we need to expand the sample size and the type of study participants to perform in-depth research. Soluble UA can inhibit IL-1 receptor antagonist promoting TLR-induced proinflammatory cytokine IL-1β production by human primary cells25. IL-1β is a key mediator of inflammatory response, which not only induces the proliferation and aggregation of neutrophils but also activates LY. Although IL-1β is critical for host response and resistance to pathogens, it also exacerbates damage during chronic disease and acute tissue injury and is associated with infection susceptibility, immune tolerance, and vaccine biology26. In view of this, soluble UA may promote an increase in the LY population through the IL-1β signaling pathway, which may contribute to host defense but may also lead to aseptic inflammation and pathological consequences. Research found that soluble UA could enhance the anti-tumor response of Th2 cells, promote antibody production by B cells, and relate to CD8+ T and NK cells27. Moreover, studies have confirmed that soluble UA as an adjuvant can play a role in the anti-tumor immunity induced by dendritic cell vaccines29. It is suggested that SUA may play an anticancer role by regulating LY when breast cancer was complicated with HUA. In the future, we plan to conduct further studies to explore the mechanism by which soluble UA promotes LY proliferation and its potential effects on the body.Clinical and epidemiological studies have shown that HUA increases the risk of developing chronic inflammatory disorders, including hypertension, acute and chronic kidney disease, obesity, metabolic syndrome, fatty liver, and diabetes mellitus+ T cell counts and IL-4 and IL-10 levels, suggesting that high levels of soluble UA may enhance the Th2-dominated immune response. Thus, physicians should assess the immune status of patients with HUA and other diseases combined with HUA by detecting LY subgroup or Th1/Th2 cytokines, in order to guide clinical treatment and rational regulation of SUA levels. Besides, soluble UA can be used as an immune adjuvant in the management of certain disease treatments, revealing a new perspective for clinical therapeutics.In conclusion, individuals with HUA exhibited increased CD414. Of the 1915 healthy participants, 274 individuals met the diagnostic criteria of HUA. All individuals included in this study did not have cancer, heart disease, diabetes mellitus, fatty liver, hypertension, gouty arthritis, or other inflammatory disease during the study period. All individuals with dysglycemia and dyslipidemia and taking antihypertensive drugs, urate-lowering drugs, diuretics, and immunosuppressants were excluded from the study. The 274 participants who were diagnosed with HUA did not have gout symptoms or gout. The general characteristics of the 1641 healthy controls (HCs) and 274 individuals with HUA included in our study are presented in Table We randomly selected 1915 healthy participants physically examined at the First Hospital of Jilin University from July 2020 to September 2020 to conduct this retrospective study. HUA is defined by SUA level of two fasting tests on different days, under a normal purine diet: males > 7 mg/dL and females > 6 mg/dLFasting venous blood was collected once in the morning. After separating the serum, the SUA concentration of all individuals was detected via the uricase-peroxidase method using a commercially available kit  and measured using a Beckman AU5841 automatic chemistry analyzer .Whole blood was used to detect the number and percentage of immune cells, including peripheral white blood cells (PWBC), LY, neutrophils (NE), and monocytes (MO). Counts and percentages were analyzed using a Sysmex XN-9000 hematology analyzer . The NE-LY ratio was determined by dividing the NE count by the LY count.Peripheral blood LY subsets of HUA patients (n = 38) and healthy people (n = 98) were tested using a FACS Canto II flow cytometer  with a BD Multitest 6-colour TBNK agent, including CD45-PerCP-Cy5.5, CD3-FITC, CD4-PE-Cy7, CD8-APC-Cy7, CD19-APC, and CD16/CD56-PE . The fluorescent beads in the absolute counting tube were used as an internal standard for the absolute counts of LY subsets. The absolute cell counts and percentages of LY subsets were analyzed using BD FACS Canto software . The gating strategy used to analyze the LY subsets is shown in Fig. The RPMI 1640 medium was prewarmed (37 °C), UA  was added, and the medium was heated to 57 °C. The final concentration of soluble UA medium was 16 mg/dL. The medium was sterilized using 0.20 μm filters. Crystals were not detectable under these conditions (as confirmed using polarizing microscopy) nor did they develop during cell incubation.g for 15 min . After washing once with phosphate-buffered saline, the PBMCs (2 × 104 per well in a 96-well plate) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and stimulated with 714, 476, 238, or 95 µmol/L soluble UA at 37 °C with 5% CO2. After 18 h of culture, the PBMCs in each well were collected. The expression of CD45, CD3, CD4, CD8, and CD19 in the PWBCs was detected via surface staining with PerCP-labelled anti-CD45, FITC-labelled anti-CD3, APC-Cy7-labelled anti-CD4, PE-Cy7-labelled anti-CD8, and Apc-labelled anti-CD19 monoclonal antibodies for 15 min, followed by flow cytometry measurements. LY was analyzed according to the gating strategy shown in Fig. In the in vitro experiments, we used Ficoll density gradient centrifugation  to separate and purify human heparinized peripheral blood and to isolate peripheral blood mononuclear cells (PBMCs). We aspirated 1000 µL of whole peripheral blood and diluted it with 1000 µL of phosphate-buffered saline. The diluted liquid was slowly dropped onto the surface of a 1500 μL Ficoll separator and centrifuged at 450×t-test or paired t-test. Differences between multiple groups were compared using one-way analysis of variance. When the data did not show normal distribution and were described as medians ± interquartile range, differences were compared using the Mann–Whitney U test. In this case, differences between multiple groups were compared using the Kruskal–Wallis test. Differences with P-values < 0.05 were considered significant. Data were analyzed using SPSS version 20  and GraphPad Prism 8 .The Kolmogorov–Smirnov test (for number of cases > 50) or Shapiro–Wilk test (for number of cases < 50) was used to assess data distribution. When the data conformed to normal distribution and were described as means ± standard deviation, differences between two groups were compared using an independent samples This study was conducted in accordance with the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of the First Hospital of Jilin University . This study was the retrospective study and the in vitro experimental study using peripheral blood lymphocytes. The in vitro experiments involve only the remaining blood samples prepared to be discarded after the use of routine laboratory tests, do not involve direct contact with the subject, and the results of the study are not used for the diagnosis of the subject. Based on this, the Ethics Committee of the First Hospital of Jilin University approved the waiver of informed consent and agreed to carry out retrospective study and in vitro experimental study. The waste samples after the in vitro study were properly disposed of according to the biological sample waste treatment method.


Cow–calf production is the foundation of the U.S. beef industry. However, infertility and sub-fertility limit its sustainability. In this review, we discuss the progress made as a result of the implementation of genomics and transcriptomics technologies to understand the genetic basis underlying fertility-related traits in cattle. We highlight the opportunities and limitations of using such technologies to discover genes and regulatory mechanisms and discuss the importance of translating research findings from bench to on-farm applications.Female fertility is the foundation of the cow–calf industry, impacting both efficiency and profitability. Reproductive failure is the primary reason why beef cows are sold in the U.S. and the cause of an estimated annual gross loss of USD 2.8 billion. In this review, we discuss the status of the genomics, transcriptomics, and systems genomics approaches currently applied to female fertility and the tools available to cow–calf producers to maximize genetic progress. We highlight the opportunities and limitations associated with using genomic and transcriptomic approaches to discover genes and regulatory mechanisms related to beef fertility. Considering the complex nature of fertility, significant advances in precision breeding will rely on holistic, multidisciplinary approaches to further advance our ability to understand, predict, and improve reproductive performance. While these technologies have advanced our knowledge, the next step is to translate research findings from bench to on-farm applications. The livestock industry is a critical component of the agricultural sector in the United States (U.S.). In 2022, cattle production was anticipated to represent about 17% of the USD 462 billion in total cash receipts for agricultural commodities ,4) to poCow–calf production is the foundation of the U.S. beef industry, representing 86% of beef operations and 84% of the beef cattle population in the country . HoweverDespite the consensus that good fertility and longevity are essential for the sustainability of the cattle industry, improving reproductive efficiency has been a major challenge worldwide ,15 due tGenomic selection (GS) has contributed to the increased productivity of yield traits and the genetic gain of low-heritability traits such as fertility, especially in dairy cattle ,24. AlthFertility is a general term encompassing a variety of traits that contribute to overall reproductive success . ReproduHistorically, selection has focused on the increased performance and growth of beef animals . EspeciaFertility is a complex, quantitative trait determined by a large number of variants with small effects that are spread throughout the genome ,35. In gOnly recently, fertility traits were included in breeding programs. The selection of fertility traits in beef cattle has its own challenges due to the diversity of breeds, production systems, and breeding goals ,38. AddiThe EPDs reported in Fertility and reproductive success are directly linked to the economics of cow–calf production operations ,44,45. DConsidering the percentages mentioned above, the selection of replacement heifers is critical to the sustainability and profitability of a cow–calf operation. Heifers are not only the genetic future of the herd but also, if the opportunity cost is factored in, they are the greatest investment beef producers can make ,42. DickEfforts have been made to identify the novel traits that need to be recorded for genetic evaluation as well as the identification and use of genetic markers through omics approaches. Novel traits such as age at calving, days to calving, first calving interval, and heifer pregnancy have been proposed for inclusion in breeding programs as they have, on average, higher heritability than previously used fertility-related traits ,49. The It is important to highlight that advances in omics technologies and analytical methods have provided opportunities to accelerate the genetic progress of low-heritability traits. These technologies can provide a snapshot of a vast amount of molecules in a tissue or cell . The priThe development of high-density genotyping assays based on SNPs (single-nucleotide polymorphisms) has led to major genetic progress in livestock species ,53,54. T® advantage test accurately predicted the heifer’s genetic merit. The Angus Australia Association, in partnership with Zoetis and CSIRO, also developed a genomic-based tool for replacement heifer selection (HeiferSELECT™) [Lately, breed associations have partnered with biotechnology companies to develop genomic tools to support GS in beef cattle ,49. LikeSELECT™) ,58. ThisSELECT™) ,40. It is essential to highlight that genomics has improved the understanding of the genetic regions modulating fertility and identified candidate genes through genome-wide association studies (GWAS). These studies have revealed thousands of quantitative trait loci (QTLs) associated with economically important traits, including fertility. NMDAR2B, SPOCK1, and ZNF462. As in dairy cattle, the GWAS studies in beef cattle have increased the accuracy of prediction for reproduction traits. Research on Brahman and Tropical Composite cattle from Australian herds showed that early reproductive performance traits had moderate to high heritabilities . Based oertility . Fortes ertility  examinedBMP6, PLEHKM3, IDH1, MAP2, and FOXO6 were among the candidate genes. It is important to highlight that although these studies have provided a better understanding of the genomic regions governing fertility, they have not yet provided causal variants or major regulatory genes. Furthermore, there is little overlap in terms of the genes or markers mentioned across multiple studies. For example, we retrieved 1483 SNPs from the CattleQTLdb associated with four fertility-related traits. As shown in Similar approaches have been used to investigate the genetic variants associated with age at first calving, heifer rebreeding, and stayability in Nelore cattle ,69,73. TAs a step forward in dissecting the molecular basis of fertility, transcriptomic studies have become a powerful tool to understand how gene expression controls cellular functions and their role in determining specific phenotypes ,76. In mThe number of transcriptomic studies is rapidly growing, as researchers have targeted different tissues, experimental models, and biological conditions to identify differences in the abundance of the genes that underlie fertility-related traits. Similarly, several tools and methods have been proposed for transcriptomics analysis . NewSOX2. This TF is involved with the reorganization of the endometrium during embryo attachment and implantation [SOX2 and OCT4 TFs as the master regulators of 773 genes associated through a GWAS with fertility in beef cattle. Under a similar experimental design, Cánovas et al. [Longissimus dorsi tissues from Brangus heifers. Among the differences in gene expression between pre- and post-pubertal heifers, the authors highlighted that most of the differentially expressed genes (DEGs) from the hypothalamus were upregulated in pubertal heifers. Additionally, the authors reported a coordinated regulation between the endocrine response of the hypothalamus and signals from the adipose tissue [Genetic merit for fertility traits affected puberty and age at pregnancy in heifers, leading to long-term effects on the performance of cows ,87. Fortantation . Neupaneantation  highlighs et al.  profilede tissue . B. indicus influenced recipient cows. The authors reported 240 P4-modulated genes that were involved in oxidative phosphorylation, biosynthetic activity, and the proliferation of epithelial cells. Furthermore, 292 genes affected by P4 negatively influenced the odds of pregnancy [Multiple factors are involved with infertility or sub-fertility in cattle, including progesterone signaling and uterine receptivity ,89. Usinregnancy . Based oregnancy . Silva eregnancy , Silva ePLIN2, TINAGL1, NPNT, LCAT, NMN, and APOA1) [Transcriptomic studies have been implemented to investigate the fetal–maternal crosstalk during the recognition of pregnancy and the interplay of P4 on conceptus elongation ,94,95. Td APOA1) ,90. SimiMNS1 is the only gene overlapping both studies  and Moorey et al. [CSF3R, NLRP1, and ENSBTAG00000049346) at breeding . However, only TFF2 displayed the same direction of expression (upregulated in non-pregnant heifers). Over-represented immune-related pathways were reported in all the studies [We overlapped the DEGs identified in publications on the PWBC transcriptome, and only a few of them were shared . Like thy et al.   or genes in a different locus or chromosome  [Genomics and transcriptomics approaches have been successful in identifying genetic variants and genes to untangle the genetic architecture of fertility-related traits. However, most of the associated variants are harbored in non-coding or intergenic regions . This su > 1 Mb) ,111,112.cis-eQTLs) regulating CCNB1 and ROR1 genes in donor cows. Likewise, they reported two other candidate genes (RARRES1 and EMC9) affected by 18 eQTLs [CCNB1 and ODF2L genes were associated with average blastocyst rate, while EMC9, MANBA, and PI16 were related to endometrial receptivity [A few studies have been performed to identify the eQTLs associated with fertility in cattle. Kadarmideen and Mazzoni  evaluate18 eQTLs . These eeptivity . p-value ≤ 10−5). Among the intervals containing eQTL and QTL for fertility, the genes FAM184B, CDAN1, and SSTR1 were identified from white blood cells [PLABG1 and CHCHD7) and oocyte competence (ATP6V1C1) [Using different approaches to identify eQTLs associated with fertility  in dairy cattle, van den Berg et al.  reportedod cells . Forutanod cells  also conod cells . As prevTP6V1C1) .This review showcases the advances that have resulted from implementing genomics and transcriptomics approaches to understand the genetic basis underlying fertility-related traits in cattle. Despite the progress made, genomic selection in beef cattle has been adopted much less than in dairy cattle . CommercWhile we can profile the transcriptome, a comprehensive understanding of the effects of the up and downregulation of differentially expressed genes is still challenging. Better analytical methods are needed to go beyond gene lists and extract meaningful information for translatable applications ,77. LikeThe lack of existing research studies implementing multi-omics and multi-tissue analyses highlights the complexity of the mechanisms governing fertility. As more data from proteomics and metabolomics approaches become available, we can expect a better understanding of the genomic regulation of fertility traits. Similarly, multi-omics data integration will allow us to discover and prioritize candidate markers. Preliminary work from our group on machine learning and network modeling has provided a framework for prioritizing genes for fertility . Approac