The use of immune checkpoint inhibitors (ICIs) targeting the T cell inhibitory pathways has revolutionized cancer treatment. However, ICIs might induce progressive atopic dermatitis (AD) by affecting T cell reactivation. The critical role of T cells in AD pathogenesis is widely known. T cell co-signaling pathways regulate T cell activation, where co-signaling molecules are essential for determining the magnitude of the T cell response to antigens. Given the increasing use of ICIs in cancer treatment, a timely overview of the role of T cell co-signaling molecules in AD is required. In this review, we emphasize the importance of these molecules involved in AD pathogenesis. We also discuss the potential of targeting T cell co-signaling pathways to treat AD and present the unresolved issues and existing limitations. A better understanding of the T cell co-signaling pathways would aid investigation of the mechanism, prognosis evaluation, and treatment of AD. AD is o+ T cells and eosinophil (Eos) infiltration into the dermis, with Eos deposition and increased Th2 cytokine expression in the skin  pathway  cell infiltrate . The infthe skin . Activat pathway . The hig pathway . Given t pathway , 14. Act pathway Box 2, P pathway –33. It iType 2 immunity is one of three types of innate and adaptive immune systems that primarily target large extracellular parasites. Alterations in type 2 immune responses are closely related to allergic diseases . Type 2  The JAK family includes JAK1–3 and tyrosine kinase 2 (TYK2), which are linked to the intracellular domains of multiple transmembrane cytokines . The JAKvia JAK1/2  can be either stimulatory or inhibitory molecules. The introduction of ICIs revolutionized the treatment of certain cancers and greatly improved survival rates. ICIs affect the balance between T cell activation and suppression by reducing inhibitory signals or enhancing stimulatory signals, thereby increasing cancer cell clearance .The unusual expression of co-stimulatory and co-inhibitory signaling pathways has been reported in various autoimmune diseases, such as systemic lupus erythematosus , rheumatIn this review, we highlight and put into perspective the current knowledge on T cell co-signaling pathways and the role of these pathways in AD Figure 12The Ig-SF is represented by the presence of Ig homology domains, which are the largest and most diverse superfamily proteins found in humans . The Ig-2.1+ T cells and 50% of CD8+ T cells in humans . Nevertheless, there was no group difference in CD28 gene expression   is applied to the ear or back of the mice, which causes an AD-like syndrome characterized by redness, scaling, swelling, scabbing, and frequent scratching. Histologically, epidermal hyperplasia and infiltration of numerous inflammatory cells are observed. This model relies on epidermal TSLP expression. The TSLP–TSLPR pathway produces Th2 cytokine-associated skin inflammation. MC903 induces CD4 lymphocyte activation, increases Eos, basophil, MC, and DC infiltration in the skin, and increases Th2 cytokine secretion.MC903 mice , 147: Th+ T cell, DC, MC, and Eos infiltration.VitD mice : VitD ca+ T and CD8+ T cell infiltration, and markedly increased Th-related chemokines in exposed skin.OVA/SEB mice : This mo+ T cell and Eos infiltration, and increased expression of the Th2 cytokines IL-4, IL-5, and IL-13. Serum OVA-specific IgG1, IgE, and IgG2a are systemically elevated.OVA mice , 144: ThHDM mice , 149: Si2.1.2CTLA4 gene contained several polymorphisms, some of which contributed to AD development in infants  mice had more severe ear swelling than PD-L1-deficient (PD-L1-/-) mice. In that study, PD-L1 was essential for attenuating Th1- and Th17A-type immunity while PD-L2 was key in reducing Th2 immunity  interacts with its ligand ICOSL (CD275) and was first reported on activated human T cells . ICOS isnd Tregs . The ICOnd Tregs .2.3.1The skin-draining lymph nodes (sdLNs) in a vitamin D3 (VitD) mouse model of AD contained increase percentages of Tregs expressing ICOS, which indicated an activated phenotype . However2.3.2+ Tregs, which had a decreased capacity for producing IL-10 when compared to Tregs from healthy controls after restimulation, while ICOS- Tregs in both groups produced very little IL-10 , similar to CTLA-4 and PD-1, is an IgSF glycoprotein with two immunoreceptor tyrosine-based inhibitory motifs , 178. BT2.4.1in vitro cellular study, human CXCR5hiPD1hi CD4 naive T (T follicular helper cells [Tfh] characteristics) were found to express elevated BTLA protein and mRNA after co-culture with thymic stromal lymphopoietin (TSLP)-activated DCs , signaling lymphocytic activation molecule 1 , SLAMF2 (CD48), SLAMF3 , SLAMF4 , SLAMF5 (CD84), SLAMF6 , SLAMF7 , SLAMF8 (BLAME), and SLAMF9  , 182. Se2.5.1Staphylococcus aureus (SA) exotoxins enhanced CD48 expression in Eos when bound to CD48, causing Eos activation and signal transduction (-/-) mice, which could be used to treat allergy . In chronic AD models (induced with 3-week OVA/SEB), 2B4-/- mice had hyperdegranulated MCs, which confirmed the inhibitory 2B4 effect on MC activation  , where bAll of the above studies, where CD2, CD48, and SLAM were blocked, described favorable effects on AD. Therefore, the CD2 family might have more co-stimulatory than co-inhibitory functions in AD. However, as the functions of the CD2 family are not entirely clear, further investigations are required to identify the best possible means of exploiting them as an AD treatment. More evidence for CD2 family functions should be obtained before it can be concluded whether these molecules can be a potential target for treating AD.2.6+ T cells, CD8+ T cells, and Tregs in both mice and humans  is a poliovirus receptor-like (PVRL) protein that belongs to the CD226 subfamily . TIGIT id humans , 193. Thd humans .2.6.1+ T cells, specifically effector memory T cells and Tregs, demonstrated enhanced TIGIT expression on the patients’ PBMCs and that the proportion of TIGIT+ cells was correlated with AD severity . In this review, we focus on TIM1, TIM3, and TIM4. TIM1 is typically expressed on Th2 cells, MCs, B cells, and NK cells while TIM4 is expressed on DCs, macrophages, and B cells  family comprises eight genes in mice, whereas only three TIM genes have been identified in humans  member of the Ig-SF, the co-inhibitory molecule CD160 is mainly expressed on T cells, NK cells, and all intraepithelial lymphocytes , 201. CD2.8.1+ T cell subset that infiltrated the inflammatory skin lesions of AD. The study also demonstrated that the engagement of CD160 enhanced the CD4+CD160+ cell proliferation induced by CD3 stimulation in ex vivo cultured T lymphocytes that infiltrated AD skin lesions  revealed that the TNF superfamily is composed of several subfamilies, of which the type V (the divergent ligands [V-THD]) and L  families members are primarily associated with co-stimulatory function, while some type L family members contribute to processes other than co-signaling functions , 204. TNin vivo . Researc3.1+ T cells and CD8+ T cells (in vivo mediator of TSLP-mediated Th2 responses (OX40 (TNFRSF4 or CD134) is a member of the type V co-stimulation family and is mainly expressed on activated CD4 T cells . The OX4 T cells . OX40 li T cells . OX40 bi T cells . OX40–OX T cells . OX40L wesponses , 209.3.1.1in vitro cell study suggested that OX40L is TSLP-induced molecule on DCs that triggers inflammatory Th2 differentiation in the absence of IL-12  is an anti‐OX40L mAb that prevents persistent inflammation . In a clPhase II clinical trials targeting the OX40–OX40L signaling pathway have achieved promising efficacy and safety results. Clinical studies in the future should verify the long-term safety and durability of these drugs.3.2+ and CD8+ T cells, which results in the establishment of T cell immunity , although both dermal DCs and keratinocytes (KCs) contributed to the inflammatory phenotype. In the absence of LCs, these cells were unable to provoke AD disease alone . That st3.2.2in vitro T cell culture study of AD patients determined that circulating Fel d 1-specific DRB1*0101-restricted CD4+ T cells expressed high levels of CD27, CD28, CCR7, and CD62L and correspondingly expressed low levels of tissue-specific homing receptors and Th1/2 cytokine production. Such CD4+ T cells with a central memory subgroup may be closely associated with long-term antigen recognition and persistent disease  is mainly expressed on macrophages and DCs (CD30 (TNFRSF8) is expressed by activated CD4 and DCs . CD30–CD and DCs .3.3.1+ cells) levels in AD patients than in non-AD controls  is a member of the TNF family of ligands that binds three distinct members of the TNFR family: HVEM, lymphotoxin β receptor (LTβR), and soluble decoy receptor 3 (DcR3) . LIGHT iture DCs –220. LIGture DCs , 222. Inture DCs , 224.3.4.1flox/flox (a specific lack of HVEM expression in KCs) mice exhibited minimal clinical symptoms characteristic of AD (AD-like dermatitis sensitized with HDM [house dust mite] antigens combined with SEB)  . In vitrrevented . Additiorevented . Importarevented .3.4.2The peripheral serum of AD patients contained significantly elevated soluble HVEM (sHVEM) and LIGHT levels . Kotani These above results suggested that reagents that target either LIGHT–HVEM interactions alone or LIGHT interactions with both of its ligands may be beneficial for therapies halting and potentially abrogating AD in humans.3.5CD40 (TNFRSF5) is a co-stimulatory molecule that belongs to the type L family. Many APCs, such as DCs and B cells, express CD40 constitutively . The CD43.5.1+ cells in the skin lesions and peripheral blood of AD patients and identified such cells as a positive correlation with disease severity , 310-nm and 340-nm ultraviolet light-emitting diodes (UV-LEDs) effectively inhibited STAT1 signaling and suppressed inflammation by downregulating ICAM-1. That study suggested that 310-nm and 340-nm UV-LED phototherapy may be a therapeutic strategy for AD  treatment was 62.9%, where most irAEs were of mild to moderate intensity . Severe Targeting treatment at co-signaling molecules may be a promising future approach. More large-sample, long-term clinical studies would aid understanding of their safety.6Co-stimulatory and co-inhibitory receptors play key roles in T cell biology and act as secondary signals to determine the functional outcome of TCR signaling. The classical definition of T cell co-stimulation continues to evolve through the identification of novel co-stimulatory and co-inhibitory receptors, the biochemical characterization of their downstream signaling events, and the characterization of their immune function. After decades of development, Chen et al. reported that co-stimulatory and co-inhibitory receptors exhibit highly diverse and complex expression, structure, and function, and their function is largely influenced by the environment . The curSeveral co-stimulatory signaling targeting drugs mentioned in this review are in the clinical research stage. The drugs  targeting the OX40–OX40L co-stimulatory pathways are clinically used in AD treatment –90. Phasin vitro cell experiments. Among them, targeting the CD40–CD40L signaling pathway is noteworthy. Conventionally, it is believed that blocking the co-stimulatory signaling pathway weakens the co-stimulatory effect and reduces the inflammatory indicators. However, strong lymphocyte proliferation and lgE expression expansion were observed in vitro cell experiments . An in-depth study of the specific mechanism is crucial for developing and improving AD targeted therapy strategies in the future. Moreover, whether there are more co-stimulatory and co-inhibitory receptors or new receptors or ligands of known molecules to be discovered cannot be ruled out, and there are more aspects worth exploring.Efalizumab was discontinued in psoriasis treatment due to adverse effects. Therefore, how can the adverse effects caused by targeting signaling pathways be minimized or even eliminated? While therapeutics using targeting co-signaling molecules offer desirable outcomes, they also present unique challenges. Can more specific targeted therapies that only act on the skin be developed? Fortunately, specific antibodies can be targeted to specific tissues and organs through the targeted delivery of engineered viral vectors or nanoparticles , 242. ItIn this article, we repeatedly mentioned combined treatment methods, such as anti-OX40L and anti-IL-4 combined, which achieved good results. Moreover, the synergistic inhibition of anti-PD-1 and anti-TIGIT yielded the best results for inhibiting T cell activity . HoweverGiven that there are no guidelines currently available, perhaps the development of synergistic signaling pathway-targeted therapies in the future can enable greater selectivity in AD treatment guidelines. Furthermore, how can specific immune cell populations be targeted more precisely? As co-signaling molecules are widely expressed on various immune cell types, targeting any pathway can yield unexpected results, such as the aforementioned biased results of the anti-CD40–CD40L signaling pathway, which is both the appeal and problem for the immune system. This raises the question of whether it might be possible to develop treatments that target a specific cell type and lead to more precise treatment and resolve the recurring episodes of AD that have long troubled doctors and patients.7This review not only discusses the current research progress of targeted therapy of co-signaling molecules in AD but also presents unresolved issues and existing limitations. Several biologics targeting co-signaling molecules have demonstrated promising results in AD patients. Blockade of the T cell co-stimulatory pathways may be a promising therapeutic target in AD, and inhibiting the T cell co-suppressive signaling pathways may produce irAEs. Given the significant impact of the co-stimulatory and co-inhibitory pathways in AD, there are plausible reasons and a clear motivation to expand our understanding of these co-signaling molecules in AD and determine their full effect. We believe that targeting the common signaling pathways for AD shows strong promise.CZ wrote the manuscript; YS and YZ critically revised the manuscript. All authors contributed to the article and approved the submitted version.


The field of antiviral therapeutics is fixated on COVID19 and rightly so as the fatalities at the height of the pandemic in the United States were almost 1,000,000 in a twelve month period spanning parts of 2020/2021. A coronavirus called SARS–CoV2 is the causative virus. Development of a vaccine through molecular biology approaches with mRNA as the inducer of virus spike protein has played a major role in driving down mortality and morbidity. Antivirals have been of marginal value in established infections at the level of hospitalization. Thus, the current focus is on early symptomatic infection of about the first five days. The Pfizer drug paxlovid which is composed of nirmatrelvir, a peptidomimetic protease inhibitor of SARS–CoV2 Mpro enzyme, and ritonavir to retard degradation of nirmatrelvir, is the current FDA recommended treatment of early COVID19. There is no evidence of broad antiviral activity of paxlovid against other diverse viruses such as the influenza virus, poxviruses, as well as a host of respiratory viruses. Although type I interferons (IFNs) are effective against SARS–CoV2 in cell cultures and in early COVID19 infections, they have not been broadly recommended as therapeutics for COVID19. We have developed stable peptidomimetics of both types I and II IFNs based on our noncanonical model of IFN signaling involving the C-terminus of the IFNs. We have also identified two members of intracellular checkpoint inhibitors called suppressors of cytokine signaling (SOCS), SOCS1 and SOCS3 (SOCS1/3), and shown that they are virus induced intrinsic virulence proteins with activity against IFN signaling enzymes JAK2 and TYK2. We developed a peptidomimetic antagonist, based on JAK2 activation loop, against SOCS1/3 and showed that it synergizes with the IFN mimetics for potent broad spectrum antiviral activity without the toxicity of intact IFN molecules. IFN mimetics and the SOCS1/3 antagonist should have an advantage over currently used antivirals in terms of safety and potency against a broad spectrum of viruses. There is a somewhat Pavlovian response to viral infections that revolves around a very specific approach to development of or identification of antivirals. This approach resides in the chemistry/pharmacology subdiscipline known as medicinal chemistry. Further, there is a penchant for looking for therapeutics via an off-the-shelf approach. Case in point; remdesivir, the broad spectrum pro drug of a nucleotide analog, was approved early by the FDA for treatment of COVID19 in hospitalized patients . SARS-Co4-hydroxycytidine which has been shown to induce mutations in the DNA of mammalian cells  family of cytokines –15. The Recently, monkeypox, a cousin to the smallpox virus and endemic to West Africa, has been reported virtually worldwide . MortaliIntrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) in proteins involved in signal transduction have been revealed primarily through the technology of nuclear magnetic resonance (NMR) –31. ThisOur peptide approach to structure/function predated the discovery that proteins involved in signaling are IDPs and/or proteins with critical IDRs. Our initial approach to studying IFNs via short peptides involved the synthesis of a 20-amino acid peptide corresponding to the N-terminus of human IFNγ (huIFNγ), coupling it to keyhole limpet hemocyanin macroprotein, and using it as an immunogen in rabbits . The resThe IFNγ and type I IFN mimetics that have resulted from the model and which are contained in the C-terminus sequences of the IFNs are presented in 382RKIIEKKT of the precursor form of human IFNAR1  results in 100 percent death by day 10. Administration of 20 mg/kg of huIFNα1 aa (152 -189) peptide mimetic I.P. up to 2 days post infection completely protected mice against death , was similarly tested in the vaccinia virus mouse model as well as cell culture . ConditiIFNγ mimetic muIFNγ aa (95-133) was also active against a highly lethal strain of the picornavirus encephalomyocarditis (EMC) virus in a mouse model with a lethality similar to that of vaccinia virus . The typsuppressors of cytokine signaling (SOCS) family. The intracellular SOCS of interest are called SOCS1 and SOCS3  virus, herpes simplex virus, influenza virus, as well as coronaviruses including SARS-CoV2 (COVID19) and OC43 that causes common cold , 54, 55.Staphylococcus aureus (MRSA) mouse model where skin infection was associated with increased SOCS1 (It has recently been reported that individuals immunocompromised with active AIDS are prone to severe monkeypox infection . Relateded SOCS1 . TreatmeWhile the paxlovid protease inhibitor nirmatrelvir is currently the FDA preferred therapeutic for early mild COVID19, a phenomenon called rebound COVID19 has been reported in paxlovid treated patients . This coWe have discovered and developed a noncanonical model of the mechanism of action of types I and II IFNs. We have further shown a remarkable similarity of this mechanism to that of steroid/steroid receptor signaling. In this regard, we have unified ligand/membrane receptor signaling with that of cytoplasmic/nuclear receptor signaling. Membrane receptor signaling is conceptually dominated by receptor tyrosine kinases and receptor associated tyrosine kinases. The latter in turn is dominated by the canonical model of JAK/STAT signaling pathway. It is our contention that JAK/STAT signaling in the context of this model is incomplete and replete with inexplicable add-on adjustments that have led to such phrases as “The STAT specificity paradox” . The cenIt is our view that their therapeutic value is hiding in plain sight, waiting to be exploited.HJ designed the research and wrote the manuscript. CA carried out the experiments. All authors contributed to the article and approved the submitted version.


STJE had significant negative correlations with CAIT , and significant positive correlations with AIIT . The cut-off value to distinguish between the CAI and asymptomatic groups was 1.7 mm using the ROC curve.The purpose of this study was to develop the assessment of subtalar joint instability with chronic ankle instability (CAI) using ultrasonography. Forty-six patients with anterior talofibular ligament (ATFL) abnormalities and a history of ankle sprain were divided into CAI  and asymptomatic groups  on the basis of subjective ankle instability assessed using the CAIT and the Ankle Instability Instrument Tool (AIIT). Twenty-six age-matched feet participated in a control group . Ultrasound measurements of the width of the posterior subtalar joint facet were obtained at rest and maximum ankle inversion . The differences in STJE among the three groups were assessed by one-way ANOVA. The relationship between STJE and subjective ankle instability was assessed using Spearman’s correlation tests. The STJE value was significantly greater in the CAI group (2.3 ± 0.8 mm) than in the asymptomatic (1.0 ±0.4 mm) and control groups (0.8 ±0.2 mm) ( Chronic ankle instability (CAI) due to repeated ankle sprains causes various dysfunctions  and decrStress radiography or MRI is mainly used for the diagnosis of STJ instability . The STJUltrasonography (US) is an imaging modality that can assess joint dynamics non-invasively and in real time. In recent years, assessment of joint instability by ultrasonography has been established for some joints ,11. For The study was approved by the Medical Ethics Committee of the authors’ institution (2016–072), and all the subjects provided informed consent for study participation. Forty-six feet of forty-six patients were included in this study. All patients had been diagnosed with a lateral ankle sprain and had undergone physical therapy at one orthopedic clinic from April 2018 to August 2019. The inclusion criteria were a past medical history of several ankle sprains and more than 1 month had passed since the most recent lateral ankle sprain at the time of testing. The exclusion criteria were having tenderness at the ATFL and/or motion pain at maximum inversion stress. Cases with tenderness in the ATFL and/ or motion pain at maximum inversion stress were excluded as there may be some residual abnormality, such as chronic inflammation of the ATFL.The structure of the ATFL was assessed using US , and subUS was performed using B mode with the EUB-7500 linear probe . Assessment of the ATFL by US was performed according to the study of Hua et al.  ATFL injThe STJ was examined at rest on a bed with approximately 20 degrees of ankle plantarflexion. The linear probe was placed at the antero-inferior aspect of the lateral malleolus. The proximal side of the probe was then attached, and its distal side was rotated backward 30 degrees to capture the STJ . The pos95) was calculated from Equation (1).Nine feet of nine subjects  who did not have any ankle instability participated in this study to examine the reliability of the assessment of STJE. Their mean age was 26.0 ± 2.2 years, height was 165.0 ± 6.1 cm, and weight was 58.2 ± 7.4 kg. Subjects with a past medical history of a sprained ankle within the past month or a past medical history of ankle surgery were excluded. The ultrasound measurements were performed by two testers, one of whom was experienced in US assessment and the other a novice assessor. The experienced tester had 5 years of experience in assessing muscles using ultrasound imaging. Training of the novice rater included basic education in ultrasound imaging techniques and practicing capturing ultrasound images under the observation of the experienced operator with instruction in the measurement technique for 15–30 min/day for 1 month. The two examiners performed assessments three times separately on the same day as the first session, and one examiner performed repeat evaluations again three times on another day as the second session. For examination of intra-rater reliability, ICC  and the standard error of the mean (SEM) were calculated from the mean value of the first and second sessions. For inter-rater reliability, ICC  and the SEM were calculated from the mean value of the first session of the two raters. Minimum detectable change 95  was used to compare the age, height, and weight of the three groups. Sex and affected side distribution were compared using the chi-squared test. The STJE, the CAIT, and AII were compared among the three groups by ANOVA with Tukey’s test as a post hoc test. Spearman’s correlation tests were performed, with calculation of the correlation coefficients of STJ rest and inversion and STJE with subjective ankle instability based on the CAIT and the AIIT.p < 0.05 considered significant.The diagnostic performance of the STJE was determined using receiver operating characteristic (ROC) curves. Putative cut-off values for STJE to calculate sensitivity and specificity were calculated. The Youden index was used to determine the best cut-off value. The STJE value corresponding to the highest Youden index was used to divide the patients into asymptomatic and CAI subgroups. All statistical tests were performed using SPSS statistics, version 25.0 , with The CAI, asymptomatic, and control groups consisted of 25 persons, 21 persons, and 26 persons, respectively. There were no significant differences among the three groups in age, height, weight, sex, and affected side. CAIT were significant differences among the three groups . AIIT were significant differences among the three groups  .95 are shown in All results demonstrated high reliability. Intra-rater reliability within the same day was 0.85 (0.64–0.96). Inter-rater reliability was 0.95 (0.81–0.98). The ICC, SEM, and MDCp-value of Control-CAI: 0.06, Control-asymptomatic: 0.13, asymptomatic-CAI: 0.90). STJ inversion showed a significant difference among the groups . The STJE value was significantly greater in the CAI group (2.3 ± 0.8 mm) than in the asymptomatic group (1.0 ± 0.4 mm) and control group (0.8 ±0.2 mm) .The outcomes of US imaging of the STJ are shown in p < 0.01, STJE: r = −0.71, p < 0.01), and significant positive correlations with AIIT .Correlation coefficient values are shown in The ROC curve used to examine the diagnostic performance of STJE as a predictor of CAI had an area under the curve of 0.94 (95% CI 0.87–1.00). The highest Youden index was 0.79, which corresponded to a cut-off value of 1.7 mm .95 value of STJ inversion, but the SEM and MDC95 values of STJ inversion were larger than those of STJE. Moreover, the correlation coefficient value of STJE and subjective ankle instability was stronger than that of the STJ width in inversion. Additionally, the cut-off value to distinguish between the CAI and asymptomatic groups was 1.7 mm. Therefore, increased STJE was one of the important factors related to subjective ankle instability.The purpose of this study was to develop ultrasound assessment of the STJ instability in subjects with CAI. The STJ width in inversion and STJE are different among the three groups. The difference in STJ width in inversion in the CAI group with other groups was greater than the MDCIn the previous study, STJ assessment did not satisfy the need for a gold standard. Thus, the relationship between the STJ instability and the pathophysiology of CAI had not been clear. Renstrom reported  that STJInjuries of the ligaments of the STJ most often occur in conjunction with a lateral ankle sprain . The excSprains of the STJ were difficult to define and were even more difficult to diagnose, because of the unclear assessment of the STJ instability. All subjects of both the CAI and asymptomatic groups had some abnormality of the ATFL and a past medical history of multiple ankle sprains. Thus, CAI patients had a past medical history of multiple ankle sprains, an abnormal ATFL, and STJE greater than 1.7 mm. We suggest that those ligament structures of patients with greater STJE require detailed assessment by magnetic resonance imaging (MRI). When these diagnostic strategies for STJ instability become established, how much the STJ instability affects the symptoms of ankle instability will be clarified. Recently, research has focused on the sensorimotor alterations in CAI. In assessing the effects of CAI on balance and other performance indicators, it is often discussed that the contralateral limb may perform similarly worse compared to healthy subjects, leading to the theory of altered sensorimotor control in CAI that affects both limbs ,28. The This study had some limitations. The STJ movement was classified as inversion-eversion around a single oblique axis inclined an average of 42 degrees in the sagittal plane and medial deviation of 23 degrees in the axial plane related to the longitudinal axis of the foot. In the open kinetic chain, supination consists of plantarflexion, inversion, and internal rotation , becauseIn the present study, inversion stress was applied manually, and the reliability of STJE was only assessed in normal volunteers. Thus, all patients were assessed by a single examiner, but the degree of manual stress might affect the STJE value.The criteria of the IAC could not be applied to this study. According to the criteria of the International Ankle Consortium (IAC) , CAI is The CFL is known to be integral in preventing excessive inversion and external rotation of the STJ ,33. CFL The most important limitation of this study was that there was no reference test. However, subjects with subjective instability had increased STJE values; therefore, it could be said that they had the STJ instability during inversion of the ankle using STJE values. Further studies are needed to investigate the relationship between the development of CAI and CFL function using MRI.The subtalar joint instability is known to be one of the factors affecting CAI; however, there were no methods to assess it in clinical settings. The STJE during inversion of the ankle could be assessed non-invasively by US. Individuals with CAI had increased STJE that was associated with subjective ankle instability. The STJE may be reflected in the subtalar joint instability, therefore it was necessary to assess not only the ATFL but also the STJ excursion in the subject with CAI. However, CAI is affected by multiple factors, and assessment of the CAI is performed in multiple dimensions, which are instability of the ligamentum structure, muscle strength, motor control, and sensory–motor function. In further studies, clarified pathophysiology may be needed to assess between several factors and subtalar joint instability.


As cell signaling research has advanced, it has become clearer that signal transduction has complex spatiotemporal regulation that goes beyond foundational linear transduction models. Several technologies have enabled these discoveries, including fluorescent biosensors designed to report live biochemical signaling events. As genetically encoded and live-cell compatible tools, fluorescent biosensors are well suited to address diverse cell signaling questions across different spatial scales of regulation. In this review, methods of examining spatial signaling regulation and the design of fluorescent biosensors are introduced. Then, recent biosensor developments that illuminate the importance of spatial regulation in cell signaling are highlighted at several scales, including membranes and organelles, molecular assemblies, and cell/tissue heterogeneity. In closing, perspectives on how fluorescent biosensors will continue enhancing cell signaling research are discussed. Cell signaling broadly describes how cells process and transmit information originating from the extracellular or intracellular environment. When the field of cell signaling was initially established, signal transduction was depicted as an essentially linear process: An external signal received at the cell surface is transduced intracellularly through a cascade of second messengers and/or other enzymes that activate effector proteins to drive a cellular response. This view typically assumed that signaling molecules are homogeneously distributed inside cells and that each distinct signal is transduced along a dedicated pathway. These early studies were instrumental in building our knowledge and laying the foundation for our understanding of signaling pathways. However, empirical observations have since demonstrated this initial, simplified model is insufficient to comprehensively describe how signaling pathways achieve specific control of cellular functions, prompting new models of spatially regulated signaling networks . AccordiThe spatial regulation of cell signaling is exerted at multiple levels. At a subcellular level, signaling pathway activity can vary greatly depending on whether pathway components are located within or on the surface of membrane-bound compartments as opposed to the cytosol. Signaling machinery can also be organized through molecular assembly to form signaling complexes with distinct activities at specific locations in the cell, helping facilitate the spatially confined production or destruction of signaling molecules by local regulatory processes, known as regulatory fencing. Finally, signaling activity can vary among cell populations and across tissues as a result of paracrine and/or endocrine effects. These mechanisms of spatial regulation ensure efficient cellular activities and facilitate acutely controlled signaling with high specificity. Loss of spatial regulation contributes to dysregulated signaling that leads to disease development . TherefoResearchers currently access numerous techniques to unravel the spatial regulation of cell signaling. Traditional biochemical techniques like cell fractionation and Western blotting remain staples of signaling research but involve lysing large numbers of cells to characterize a subset of known molecules, which limits spatiotemporal resolution and can obscure potentially important single-cell behaviors. Multiple ‘omics' technologies are also greatly contributing to our understanding of cell signaling by enabling high-throughput characterization of an array of molecules at discrete time points . ProteomAequorea victoria green fluorescent protein (GFP) into a rainbow of fluorescent protein (FP) variants transformed the study of cell signaling by enabling the development of genetically encoded fluorescent biosensors [−) [The Nobel Prize-winning discovery and engineering of osensors . The bassors [−) . A more sors [−) . Biosenssors [−) . The senAmong the simplest sensor configurations are translocation-based biosensors, in which a specific signaling protein or protein domain is fused to an FP and used to indicate the accumulation and/or distribution of a target molecule based on the redistribution of the fluorescence signal from one part of the cell to another. Many phosphoinositide biosensors feature this design, attaching FPs to different lipid-binding domains (e.g. pleckstrin homology domains) . Other tBy far the largest and most successful class of biosensors are switch-engineered biosensors. In this design, the sensing unit acts as a molecular switch and undergoes a conformational change upon detection of a biochemical event, which then modulates the fluorescence of the reporting unit. A molecular switch can be directly incorporated into an FP β-barrel, such that conformational changes will distort the FP structure to alter the chromophore environment and, therefore, FP photophysical properties  ,24. ThesSuch single-fluorophore switch-engineered biosensors are becoming increasingly popular for their high sensitivity and compatibility with multiplexed biosensor imaging . HoweverBeing genetically encodable means that fluorescent biosensors can be constructed using standard molecular biology techniques and introduced to cells as DNA, whereby the cell's own protein synthesis machinery generates the biosensor(s). Genetic encodability also offers the opportunity for enhanced spatial selectivity, as one can harness native cellular machinery to tailor biosensor localization to match the spatial scale of the biological question . For exaParallel technological advancements in molecular engineering and microscopy have enabled researchers to go beyond initial theories of linearized cell signaling to directly visualize previously undiscovered aspects of spatial regulation in cell signaling. Within the scope of this review, we highlight recent biosensor developments that reveal the spatial regulation of cell signaling at the level of membranes and organelles, molecular assemblies, or cell/tissue heterogeneity.Membrane-bound organelles represent enclosed signaling compartments whose physical boundaries sequester specific biochemical processes while excluding others. The surfaces of lipid membranes can also serve as signaling platforms with distinct properties from those of the bulk cytosolic environment. Biosensors can be directly targeted to different subcellular compartments through the inclusion of sequences encoding endogenous organelle-localization signals or by fusing a biosensor to a protein known to be enriched on or in a given organelle.Understanding the regulation of metabolic activities across organelles is a natural fit for this approach, as individual organelles engage in distinct catabolic and anabolic processes to respectively break down or build up the intracellular and extracellular environment ,40. AnabThe targeting of phosphoinositide-, Akt-, and mTORC1-specific biosensors has identified specific lysosomal regulation in PI3K signaling. Lysosomes have emerged to be a functionally important site for the Akt–mTORC1 pathway ,47. Howe3 and PIP2) are present on the lysosomal membrane to recruit Akt, using an indicator for phosphoinositides based on Akt (InPAkt). This biosensor contains a molecular switch comprising the pleckstrin homology (PH) domain of Akt linked to a negatively charged pseudoligand peptide and exhibits a C/Y FRET change upon displacement of the pseudoligand by the binding of PIP3 or PIP2 to the PH domain [Having detected Akt on the lysosomal membrane by expansion microscopy, a super-resolution method that involves sample expansion , Chen etH domain . InPAkt H domain . Indeed,H domain . OverallBeyond lysosomal PI3K signaling, studies using targeted fluorescent biosensors have also identified distinct Akt/mTORC1 regulation in the nucleus. From the earliest observations of Akt and mTORC1 in nuclear fractions of cell lysates, multiple groups have been attracted to the potential of Akt/mTORC1-mediated transcriptional regulation controlling survival, protein synthesis, and the metabolic capacities of the cell, especially considering the hyperactivation of Akt and mTORC1 in some cancers . StudiesTo approach this question, Zhou et al.  developeActing opposite mTORC1-directed anabolic signaling, adenosine monophosphate (AMP)-activated kinase (AMPK) regulates distinct catabolic processes across the cell to promote energy production . AMPK stEarly C/Y-FRET-based AMPK biosensors confirmed that metabolic stress activates AMPK in the cytoplasm and at the plasma membrane, as well as on the Golgi, ER, mitochondrial, and lysosomal surfaces ,71. One Indeed, recent work targeting an improved AMPK biosensor to various organelles has revealed distinct compartment-specific signaling kinetics, including isoform-specific and upstream-kinase-dependent effects. Using the sensing components found in previous C/Y-FRET AMPK biosensors, Schmitt et al.  were ablWhile previous investigations did not show meaningful nuclear AMPK activation in response to metabolic stress, ExRai AMPKAR facilitated robust detection of LKB1-induced nuclear AMPK activity in response to metabolic stress, as well as through direct allosteric activation. To clarify the regulation of nuclear AMPK activity, the ability of nuclear-localized or diffusible AMPKα2 to promote nuclear AMPK activity in AMPKα knockout cells was compared, demonstrating that only diffusible AMPKα2 was able to drive nuclear AMPK activity. These results helped formulate a mechanistic model of nuclear AMPK activity in which AMPK is first activated in the cytosol and then translocates into the nucleus to phosphorylate downstream targets. This model and associated methods of studying nuclear AMPK translocation and activity are particularly useful, as downstream AMPK targets that are exclusively nuclear, rather than fellow shuttling components, are under active investigation . ApplyinCarefully executed organelle- and membrane-targeting approaches can even upend established signaling paradigms, as is the case with a recent study of GPCR-mediated activation of extracellular regulated kinase (ERK) signaling. As GPCRs are an abundant family of signaling receptors targeted by over a third of existing small-molecule drugs, it is critical to understand the molecular mechanisms of GPCR signal transduction . As theiUsing the well-studied β2 adrenergic receptor (β2AR) as a model, Kwon et al.  sought tBy targeting these molecular tools to either endosomes or the nucleus, the authors sought to manipulate β2AR signaling in a location-specific manner. Responses from endosome- and nuclear (NLS)-targeted EKAR4 demonstrated that active, endosome-localized β2AR signaling machinery is required for ERK activity at both endosomes and the nucleus. In particular, biochemical experiments suggested that active, endosome-localized Gαs directly recruits the upstream components RAF1 and MEK1 to initiate endosomal ERK activity. These results delivered a new model of non-canonical GPCR-mediated ERK signaling where β-arrestin facilitates GPCR endocytosis and endosomally localized active Gαs recruits RAF1 and MEK1 to activate ERK signaling, which ultimately propagates to the nucleus. This model showed further promise when considered in the context of myelodysplastic syndrome, which describes a group of blood cancers typified by a long splice variant of Gαs and up-regulated ERK activity ; indeed,Molecular assemblies represent micro- or nanometer-scale signaling compartments that are organized directly at the protein level. In particular, the ability of many signaling proteins to incorporate into so-called ‘membraneless organelles' has attracted intense interest in the last decade, spurring investigations into their role in the spatiotemporal regulation of intracellular signaling. These dynamic assemblies, also referred to as biomolecular condensates, form through a process known as liquid–liquid phase separation (LLPS), which describes a phenomenon whereby groups of molecules overcome the tendency to be homogenously distributed in solution and spontaneously coalesce into membraneless, liquid-like droplets that are physically distinct from the surrounding environment .in vitro and in living cells. Notably, the authors took advantage of the spontaneous fragment complementation of split superfolder GFP (sfGFP) to examine the behavior of endogenously expressed RIα. In this system, the eleventh β-strand of sfGFP (sfGFP11) and the remaining ten β-strands (sfGFP1-10) are expressed as separate fragments that show no fluorescence alone but spontaneously assemble to reconstitute fluorescent sfGFP when co-expressed [11 facilitates its insertion into endogenous genomic loci using CRISPR/Cas9 technology, so that sfGFP1–10 can be reconstituted at an endogenously expressed protein of interest (POI) [Recently, work by Zhang et al.  revealedxpressed . The smast (POI) , in thisin vitro, but that this could be reversed by adding cAMP, indicating that cAMP regulates RIα LLPS. Indeed, they also found that treatment with cAMP-elevating stimuli rapidly up-regulated the formation of RIα condensates in cells. Finally, when the authors co-expressed FP-tagged RIα and PKA-C in cells, they observed PKA-C to co-phase separate with RIα, prompting them to examine cAMP and PKA dynamics directly within these phase-separated bodies [A tetrameric holoenzyme consisting of an R subunit dimer bound to a pair of catalytic (C) subunits, PKA becomes activated when cAMP binds the R subunits and triggers an allosteric switch that unleashes PKA-C to phosphorylate downstream targets. Notably, Zhang et al.  observedd bodies .1–10 to a POI endogenously tagged with sfGFP11, FluoSTEPs pair sfGFP1-10 as a partial FRET donor with a red-emitting FRET acceptor, such that reconstitution of sfGFP produces a functional G/R FRET biosensor only at the location of the endogenously expressed POI, eliminating issues related to POI overexpression.To investigate signaling dynamics associated with molecular-scale assemblies, it is important to engineer biosensors that can probe signaling activities at a comparable spatial scale. For example, analogous to how biosensors can be targeted to organelle membranes via fusion to localization motifs, biosensors are often fused to POIs to visualize nearby signaling. Resolving signaling activities at specific molecules, however, raises concerns about the biological limitations of overexpression systems . In partFluoSTEPs closely mirror the designs of existing FRET-based biosensors and can be readily generalized to detect various signaling targets . SpecificAMP signaling is generally initiated at the plasma membrane following ligand binding to Gαs-coupled GPCRs, which activate adenylate cyclases (ACs) to produce cAMP from ATP. cAMP then binds various downstream effectors, including PKA, which regulates cell proliferation, differentiation, and multiple tissue-specific processes . With huHowever, this model of PDE-driven compartmentation is contradicted by computational studies based on early observations that cAMP diffusion is unrestricted in cells . FluoresIntriguingly, the cAMP- and PKA-enriching behavior of RIα biomolecular condensates suggested that they may in fact function as a cAMP buffering system. To investigate the possible interplay between PDEs and RIα condensates, Zhang et al.  modifiedThe cAMP-nanoruler approach pioneered by Bock et al. has also recently proven instrumental in deciphering another key question in cAMP signaling, namely, how cells are able to distinguish cAMP elevations induced by specific GPCRs. To investigate this question, Anton et al.  fused EpIn addition to cAMP compartmentation, the specificity of PKA signaling is also increased through association with A-kinase anchoring proteins (AKAPs), a group of scaffold proteins that recruit PKA into discrete molecular complexes containing numerous other signaling molecules which regulate distinct functions at varying subcellular locations ,111. OveFortunately, there are now numerous super-resolution microscopy methods that push fluorescence imaging beyond the diffraction limit. For example, photochromic stochastic optical fluctuation imaging (pcSOFI) utilizes reversibly photoswitchable FPs, whose spontaneous flickering emission behavior is used to generate images with a resolution below the diffraction limit . HoweverWith the limited options for FPs that can be utilized for super-resolution applications and the Dronpa chromophore being non-critical for FLINC, Lin et al.  subsequeWhile the previous sections focused on subcellular spatial regulation of signaling activity, we now venture into cell and tissue heterogeneity, which encapsulates variation in signaling across gap junctions as well as through paracrine and endocrine effects. Investigating this scale of signaling is critical for understanding how multi-cellular organisms function as different populations of cells execute tasks and signaling activities they are phenotypically specialized to perform. This is where signaling heterogeneity arises, as a specific group of cells may initiate or direct signaling among a larger population or within a specific physicochemical environment. Fluorescent biosensors have utility at this scale to extract information about heterogeneity in cell signaling across time, whereas alternative methods can obscure cell–cell variability or lose temporal information. If observation is only possible at a few discrete time points, the selected frequency and length of observations can hinder the observation of complex temporal behaviors.in vivo cell cycle investigations by using red-shifted FPs [Cell-to-cell heterogeneity observed in various processes may be tied to cell cycle stage variation, which affects a variety of signaling activities that cells can execute . The impfted FPs .in vitro and in vivo investigations to probe connections between cell cycle status and neural and cardiac development in embryogenesis [Since the first FUCCI was reported, these fluorescent biosensors have broadly impacted both fundamental biology and translational research, particularly in stem cell and cancer research. In studies examining the effect of cell cycle stage on cell fate during embryonic stem cell differentiation, FUCCI has been used to reveal that shorter G1 length is maintained in pluripotency, with cells differentiated in early G1 showing an endoderm fate and cells differentiated in late G1 having a neuroectoderm fate . This woogenesis . Applyinogenesis ,138. In ogenesis  found thogenesis . Servingogenesis . The impFUCCI has also proven essential for directly providing new insights into mechanisms of cell cycle regulation. Proliferating cells deprived of mitogenic stimuli are typically observed to form two cell populations: one group of cells that stops dividing and another that continues through the cell cycle . The latTo investigate this observed heterogeneity in the apparent timing of the restriction point, Cornwell et al.  recentlyIn connection with cell cycle heterogeneity, it is also unreasonable to assume metabolic synchronization among a cell population similarly facing chemical and spatial limitations on growth and survival. While there are long-standing standardized benchmarks for organism-level metabolic homeostasis, describing the underlying contributions of specific tissues or even individual cells is critical for understanding metabolic disorders and developing effective drugs ,156. WitWhile our understanding of tissue-specific metabolic functions grows with AMPK and other pathways, less is known about single-cell metabolic variations. Towards this goal, fluorescent biosensors can be used to directly probe metabolites and signal transduction elements in real time to quantify metabolic heterogeneity. In a recent study from Hung et al.  energetiFurther work using common cell lines expressing AMPKAR2 treated with various oxidative phosphorylation (OP) inhibitors over several hours identified a population of OP-independent cells with little evidence of energy stress and a population of OP-dependent cells with strong AMPK fluctuations . Using aWhile we previously discussed GPCR-mediated ERK activity, ERK is primarily activated in response to growth factor signaling, an active area of interest in cancer research as tumors hijack growth factor signaling pathways to fuel their rapid growth ,163. Iniin vivo investigation of ERK activity by Hiratsuka et al. [Fortunately, fluorescent biosensors can meet the temporal need to probe ERK cell–cell signaling heterogeneity across the entire timeframe of interest to investigate when and how cells have variable growth factor-stimulated ERK activity. Based on imaging of a C/Y-FRET based ERK activity reporter with an EV linker (EKARev) over several hours, the signaling heterogeneity was first described as ERK pulses that were frequency modulated by growth factor receptor activity and amplitude modulated by the upstream kinase MEK ,166,167.a et al.  this heta et al. . In the As several groups have continued working in this area, more factors that contribute to growth factor-based ERK signaling heterogeneity have been identified, including cell density, growth factor specificity, and further downstream kinases . While sRecent investigations of ERK heterogeneity have been focused on understanding the integration of biochemical and mechanical cues in collective cell migration, critical for understanding embryonic development, cancer cell invasion, and wound healing ,175,176.Further work by Hino et al.  describeOn a technical level, fluorescent biosensors represent highly adaptable tools to quantify signaling at different scales of spatial regulation, from the tissue-level all the way down to the nanoscale . When puin vivo performance of most traditional fluorescent biosensors, owing to the scattering and absorption of visible light by complex tissues, drives the ongoing need to adapt existing fluorescent biosensor designs to use red-shifted (e.g. far-red or near-infrared) fluorophores, as well as to increase compatibility with multiphoton microscopy methods, especially in neuroscience, for in vivoimaging. The modularity and utility of biosensors facilitates their integration with numerous technologies that are being used to improve our understanding of cell signaling in the 21st century. At the molecular scale, for example, genetically encoded fluorescent biosensors have been integrated with super-resolution microscopy techniques to illuminate nanoscale signaling events [The use of biosensors continues expanding from (1) limited signaling pathways to broader signaling pathways, (2) limited localizations to a broad spectrum of protein- and organelle-specific targeting, and (3) fewer cell/tissue types to broader adoption in a wide array of primary cells and animal tissues. For animal studies in particular, the poor g events ,121. Howg events ,182–187.g events .On a biological level, fluorescent biosensor studies continue to reveal novel insights that broadly impact pharmaceutical and medical sciences. In this review, we highlighted recent applications of biosensors to describe aspects of spatial signaling regulation that are critical in numerous areas, including metabolism, cancer, and cell development. Just as the transcriptional, translational, and post-translational regulation of signaling elements are essential to study, understanding the spatial regulation of these components provides vital information to understand fundamental biological functions. Moreover, after biosensors identify signaling mechanisms in homeostatic contexts, they can be used to directly investigate contexts of disease-related deleterious signaling and to explore the mechanism of therapeutic interventions. Fluorescent biosensors thus enable us to go beyond simplified linear models of cell signaling to describe the intricate spatiotemporal regulation of signaling networks across scales that will illuminate 21st century biomedical research.


Moderate to high heritability estimations for fertility per cycle (hs2 = 0.08) and end-of-season fertility (hes2 = 0.36) were obtained. Identification of the stallion fertility variations based on different breeding factors is useful to improve the stallion’s reproductive efficiency, to help the breeders to make the most relevant choices before the beginning of the breeding season, and to have better profitability and the sustainable use of their animals.Stallion fertility is a vast subject, with a wide array of factors that can impact reproductive performance in either positive or negative ways. To better valorize horses’ genetic resources and understand the factors affecting variations in stallion fertility per cycle and end-of-season fertility, we have performed a statistical analysis study on some breeding factors: year of breeding, stud farm, age of the stallion, number of covered mares per stallion, reproduction methods, and age of the mare using 94 purebred Arabian stallions in four different regions of Tunisia. The pertinent results are: Sidi Thabet’s stud farm contained the highest number of purebred Arabian stallions. The majority of stallions were between 15 and 21 years old and had covered 1 to 20 mares; 95.19% of the stallions were used in natural mating. Depending on the year and stud, there was a variation in the fertility per cycle and end-of-season fertility of the stallions. The use of artificial insemination with fresh semen showed a high average value for fertility per cycle and end-of-season fertility. The used statistical model showed that the breeding year, the stud, the age of the stallion, the number of covered mares by stallions, and the method of reproduction significantly affected both fertility per cycle and end-of-season fertility (with p = 0.001). Low to moderate heritability estimations for FERPCE (hs2 = 0.08) and FERPSE (hes2 = 0.36) were obtained.A consistently high level of stallion fertility plays an economically important role in modern horse breeding. To better understand the factors affecting variation in stallion fertility, we have performed a statistical analysis study on some breeding factors: year of breeding, stud farm, age of the stallion, number of covered mares per stallion, reproduction methods, and age of the mare. This work was conducted on 94 purebred Arabian stallions in four different regions of Tunisia. The results showed an increase in the number of stallions during the study period, ranging from 11.33% in 2011 to 13.29% in 2018. Sidi Thabet’s stud farm contained the highest number of purebred Arabian stallions. The majority of stallions were between 15 and 21 years old and had covered 1 to 20 mares; 95.19% of stallions were used in natural mating (Nat); 50.36% had low fertility, 17.69% had medium fertility, and 32.3% had excellent fertility according to fertility standards. Depending on the year and stud, there was a variation in fertility per cycle (FERPCE) and end-of-season fertility (FERPSE) of the stallions. The highest average FERPCE and FERPSE values were obtained using artificial insemination with fresh semen (AIF). Analysis of FERPCE and FERPSE showed that the model used in our study explained 40.21% of total variability observations for FERPCE and 42.1% for FERPSE. The used statistical model showed that the breeding year, the stud, the age of the stallion, the number of covered mares by stallions and the method of reproduction significantly affected both FERPCE and FERPSE (with  Reproductive traits are critical not only to the survival of species in natural populations but also to the survival of the livestock industry, as they have a significant impact on the economic performance of breeders and farms. This is especially important for horses, as fertility problems can have serious economic implications; mares are expensive to raise and will be a financial loss if they do not produce foals each year. Despite their importance, fertility traits are generally considered less important than performance traits in most official breeding programs.Unlike males in other agricultural species, stallions become studs based primarily on pedigree, production records, and size, regardless of reproductive health. The horse industry is full of bulls whose fertility is below the standards of other farm animals. For this reason, it should be included as a breeding objective, particularly in horses, where the trait has received less interest than other species. It is a trait that determines the productivity of breeding, and its improvement is the main objective of zoo technicians. In the last decades, many studies have focused on cattle fertility as a possible breeding goal, both in the beef industry ,2,3 and,In the horse industry, many scientific studies have dealt mainly with nutrition  and factResearchers have reported a fertility rate of around 70% to 90% in most domestic species in conjunction with new breeding techniques, whereas, with horses, the fertility rate varies between 40% and 85% . FocusinThe aim of this work is to study the effect of extrinsic factors, taking into account the effects of the region, the mating season, the number of covered mares and the method of reproduction, and intrinsic factors, such as the age of the stallion, as a contribution to a more detailed analysis of Tunisian purebred Arabian fertility stallions and to estimate their heritability. Our approach is, therefore, exclusively descriptive and is a survey-based analysis.A total of 94 purebred Arabian stallions  stationed in Tunisian national stud farms was used during the breeding season.We obtained information related to the 94 purebred Arabian stallions, collected from a consultation of the records produced by the national stud farm of Sidi Thabet and the SIRET database (Identification System Listing Equidae in Tunisia) and the data pedigrees for each stallion. For the included places of breeding (STUD) represented by Sidi Thabet, Raccada, Ben Guerdane, and Maknassy, the analyzed data were: the age of stallions and mares, the year of breeding (2011–2018), the number of mares covering each stallion, the fertilized as well as the exploited cycles, the number of empty mares, the number of mares without information (mares for which no information has been recorded in SIRET), the number of pregnant and seen mares , the dates of the covering for each stallion, the foaling dates, the covering result, the method of reproduction: natural mating (NAT), artificial insemination with fresh semen (AIF), and artificial insemination using frozen semen (AIFZ), the fathers and mothers of each stallion, the fertility per cycle (FERPC) and the end-of-season fertility (FERPS) for each stallion, father and grandfather. Fertility was calculated as follows:Fertility per cycle = (Number of fertilized cycles *)/(Number of cycles used with known result **).* The number of cycles resulting in pregnancy, including early pregnancy, followed by early loss of embryos.** The cumulative total of all cycles bred (by NAT or AI) using all mares attributed to the stallion.End-of-season fertility = (Number of pregnant mares (Fertilized cycle) + (Number of mares without information*FERPCE))/Number of covered mares.The number of mares without information reflects the number of mares that have not had a full or empty diagnosis, i.e., mares that have escaped observation (it is not known if they are full or empty).The total number of full mares at the end of the season is obtained by calculating the number of mares that have an exact diagnosis as a full mare and adding a probability of full mares among the mares without information (we do not know their exact status) multiplied by fertility per cycle.From the dates of the covering and the dates of the foaling, we calculated the intervals between foaling and first heat (IF1H), the interval between foaling and last heat (IFLH), and the interval between foaling and foaling (IFF).−The distribution of the stallions according to the year, and breeding place.−The distribution of stallions and mares according to their age.−Mares status.−Reproduction methods.−Average values of FERPCE and FERPSE. −Ranking of stallions according to fertility.−Fertility variation according to the stud farm.−Fertility variation according to the year of mating.−Fertility variation according to the reproduction methods.−Fertility evolution according to the age class of the stallions.A first descriptive step was carried out with Excel software, allowing the determination of the following:The second step was the statistical processing of the data carried out with the software SAS , with which we made a variance fertility analysis (per cycle and end-of-season) using the GLM procedure . The model for this analysis included: the effects of breeding year, breeding place (stud), the age of the stallion, the number of mares covered per stallion, the reproduction methods, and the age of the mare.The difference between means results were tested with the Duncan test.Fi: fertility (FERPCE) or FERPSE).µ: the average.Aj: jth year of breeding.Hk: kth stud farm (mating place).AGTI: Age of the stallion.NBJm: mth number of covered mares per stallion.MODEn: nth reproduction methods.AGEJo: oth age of the mare.Eijklmnop: the residual error.For a stallion, the best indicator of fertility is to calculate his FERPCE, which represents the percentage of chance of having a pregnant mare at the end of the heat.A stallion has a low FERPCE value if it is less than 48%.A stallion has a medium FERPCE value if it is between 48% and 55%.A stallion has an excellent FERPCE value if it is more than 55%.Based on their FERPCE values, stallions were ranked as having low, medium, or excellent FERPCE. A ranking was made according to the following fertility standards established by : A stallThe third step is the calculation of the heritability of a stallion’s fertility from the regression of the stallion to his sire.2 = 2 × b.To study the heritability of the stallion’s fertility, we used the regressions of the fertility of the offspring on their father, which is calculated by doubling the linear regression coefficient (b) of the fertility of the offspring for their father (bd/P), i.e., hLinear regression coefficients (b) are determined using the reg procedure on SAS.Remarkable variations in the number of purebred Arabian stallions have been noted in recent years. During the period between 2011 to 2018, we noted the use of a total number of 94 purebred Arabian stallions at national breeding stations. The selection of stallions is made according to criteria based essentially on origin, conformation, and performance. For the distribution of stallions according to the place of breeding, we noted that Sidi Thabet stud farm contained the highest number of purebred Arabian stallions (60.04%); on the other hand, the lowest number was recorded at Raccada stud farm (11.53%). This distribution was in accordance with the data reported by , showingThe high number recorded in Sidi Thabet could be explained by the importance of this stud farm in terms of accommodation capacity; it is the largest one. This increase in numbers throughout the Tunisian territory justifies the importance of this breed, which is considered among the best breeds in the world and is characterized by robustness, fertility, and its use in the various equestrian disciplines .In general, the average purebred Arabian horse’s life expectancy is around 25 to 30 years. In our study, the average age of the stallions used was around 19.24 ± 3.84 years, with a range of 6 to 30.We noticed that 0.9% of the stallions were less than 8 years old, 6.98% were between 8 and 14 years old, 73.81% were between 15 and 21 years old, 15.05% were between 22 and 28 years old, and 3.26% were over 28 years old. In summary, the majority of stallions were between 15 and 21 years old according to The average age of the mares used was 10.23 ± 5.07 years. According to We noticed that 57.32% of the mares were full, 23.69% were without information, and 18.16% were empty. The abortion rate in this study is 0.82%, while the mortality rate is 0.02% . These rMoreover, Ref.  has alsoBased on the conducted investigation, the frequency rate of covered mares by stallions varies, as shown in It is worthy of note that reproduction methods vary essentially according to the existence of the stallion in the act of reproduction. The choice of a method would depend on the availability of the chosen stallion’s semen, the choice of the breeder, the expected fertility per cycle, the rules of the different studbooks, and the cost of the technique, which must be related to the economic value of the unborn foal .In this study, the methods used for reproduction mares in Tunisian national stud farms are essentially NAT (95.19%) since the stallions are present. Artificial insemination using frozen semen (AIFZ) and artificial insemination using fresh semen (AIF) are also used with 4.61% and 0.2% of mares, respectively.Frozen semen is mainly imported and very expensive, which explains the low use of insemination using this method.The fresh semen reproduction method is conducted in the case where the stallion is very solicited due to his performance.The average IF1H is around 68.54 ± 140.21 days, with a minimum of 5 days and a maximum of 1134 days . This inThe average IFLH is around 83.58 ± 143.22 days, with a minimum of 5 days and a maximum of 1138 days.The average IFF is around 623.97 ± 297.89 days, with a minimum of 311 days and a maximum of 1489 days.The collected data explains why foalings are not regular, as mares do not give birth every year. This is mainly due to health problems and poor control of the reproductive behavior of mares, such as heat losses and the early non-use of heat occurring after foaling, such as foaling heats or the first cyclic heats. These results are very far from those found by , where tThe average father’s fertility per cycle (FERPCF) is around 45.89 ± 9.75%, with a minimum of 28.57% and a maximum of 70%.The average father’s end-of-season fertility (FERPSF) is around 65.95 ± 8.91%, with a minimum of 58.67% and a maximum of 96.33%.The average grandfather’s fertility per cycle (FERPCG) is around 45.89 (9.75)%, with a minimum of 28.57% and a maximum of 70%. The average grandfather’s end-of-season fertility (FERPSG) is around 65.95 (8.91)%, with a minimum of 58.67% and a maximum of 93.33%.In this study, 50.36% of stallions had low fertility, 17.69% had medium fertility, and 31.95% had excellent fertility.According to A variation in fertility in national stud farms was recorded . FERPCE The highest FERPCE values are recorded at the SidiThebet and Meknassy stud farms; this is mainly due to the good follow-up by ultrasound and the choice of mares at the right time, while the lowest FERPCE value is recorded at the Raccada stud farm, which lacks the convenient monitoring of mares.FERPSE reached 81.25% when using AIF, 73.51% when using AIFZ, and 70.19% when using NAT.Hence, the average values of FERPCE and FERPSE were the highest using AIF, while the average FERPCE and FERPSE values were the lowest when using NAT.Fertility using NAT is below AIF because, in this study, the used methods for reproduction mares in Tunisian national stud farms were essentially NAT (95.19%). Artificial insemination using frozen semen (AIFZ) and fresh semen (AIF) were 4.61% and 0.2%, respectively, so it is normal that we obtained the result of fertility using NAT being below AIF; additionally, the success of fertilization using fresh or frozen semen is linked to a good follow-up by ultrasound, which is practiced only in one stud farm among four farms, and is also linked to the type of semen used, whether it is fresh or frozen, which is exposed to different variation factors, in particular: collection conditions, the rhythm and frequency of semen collection, age, etc.p = 0.0001).The analysis of variance for FERPCE showed that the model explained 40.21% of the total variability of the observations; the rest (59.79%) could be explained by other factors, namely, food, the monitoring of reproduction, and the management of breeding. The statistical model used significantly affects FERPCE . We noticed that all the variations factors significantly affected FERPSE, with a probability of p = 0.0001, respectively, for the breeding year, the stud, the age of the stallion, the number of mares covered by stallions, and the method of reproduction.For FERPSE, the same statistical model and factors were used. The analysis of variance for FERPSE showed that the model explained 42.16% of the total variability of the observations. This model significantly affects FERPSE  because all the stallions used in our study received the same food ration, and therefore, we cannot talk about the effect of nutrition on fertility even if it is important.The feeding of the stallions in Tunisian national stud farms is done in two daily meals; each meal consists of 2.5 kg of barley (concentrated feed), hay (7 kg) and green grass if it is available, vitamins (if possible), and water (twice). Stallions  and foals also receive mash (2.5 kg a day/3) to increase muscle development. Watering for stallions is individual. They are taken to the troughs, one by one, two to three times a day. In the paddocks, horses have free access to drinking troughs.Heritability is a genetic parameter measuring the part attributed to genetics in the variability of performance in a given population. It is low in several species, including horses ,25,26,27Studies have reported an estimation of the heritability of stallion fertility, ranging from 0.004 to 0.15 ,33.This study reveals regression coefficients b1, equal to 0.04 for fertility per cycle, and b2, equal to 0.18 for end-of-season fertility.s2 = 0.08 for FERPCE and hes2 = 0.36 for FERPSE. This means that the additive effect genes explain 8% of fertility variability per cycle and 36% of the end-of-season fertility variability. The estimation of the heritability of FERPCE was within the range found by [Low to moderate heritability estimates for both FERPCE and FERPSE were obtained, namely, hfound by ; on the Heritabilities for reproductive parameters such as fertility are low because of the influence of environmental factors or management and the influence of dominance and epistasis. This could be caused by differences in the population structure and in the reproductive management of the different studs .Identification of stallion fertility variation based on different breeding factors is of great usefulness for stallion reproductive efficiency determination and helps the breeder to make the most relevant choices before the start of the breeding season.Due to the use of different breeding methods , knowledge of fertility heritability in breeding programs has gained increasing importance. This study has shown low to moderate heritability estimations for per-cycle and end-season fertility, respectively. The low fertility efficiency had also been partially attributed to the fact that broodmares have been traditionally selected for their war, draft, and sporting abilities rather than fertility traits.Stallion reproduction is a supervised activity that requires regulatory knowledge and technical skills that are to be carried out correctly: Mastering reproductive techniques is important to ensure good fertility results, with good gynecological and ultrasound practices to avoid losing pregnancies, and to estimate the correct time to inseminate a mare. Additionally, it is necessary to master the techniques of freezing semen to guarantee good quality semen. These results will allow the use of genetic variation and direct selection in the breeding process, as the selection of breeding parameters offers the possibility of indirectly improving animal reproductive performance and breeding station profitability, thereby increasing economic profitability.The follow-up of this study is envisaged by integrating comprehensively phenotyped Arabian stallions with genomic information in order to identify relevant candidate genes and genomic regions in correlation with fertility variations.To date, few studies have provided a better understanding of the genomic mechanisms underlying stallion fertility. This may be due to the lack of large, reliable phenotypic datasets, which hampers the modeling of environmental and managerial factors  that affect this trait. Furthermore, fertility is a complex polygenic trait with low heritability, and it is influenced by a large number of genes, with small absolute effects for each gene.


Functional neurological disorder (FND) is a common and disabling disorder, often misunderstood by clinicians. Although viewed sceptically by some, FND is a diagnosis that can be made accurately, based on positive clinical signs, with clinical features that have remained stable for over 100 years. Despite some progress in the last decade, people with FND continue to suffer subtle and overt forms of discrimination by clinicians, researchers and the public. There is abundant evidence that disorders perceived as primarily affecting women are neglected in healthcare and medical research, and the course of FND mirrors this neglect. We outline the reasons why FND is a feminist issue, incorporating historical and contemporary clinical, research and social perspectives. We call for parity for FND in medical education, research and clinical service development so that people affected by FND can receive the care they need. Some women get erased a little at a time, some all at once. Some reappear.—Rebecca SolnitFollowing centuries of neglect, gender and sex discrimination in healthcare now receives more widespread attention; but despite improved awareness, inequality remains common in biomedical settings. Research funding is disproportionately directed towards the investigation of diseases that primarily affect men at the expense of those that affect women.The impact of implicit biases on the basis of sex and gender can be seen in the lack of recognition of marginalised, stereotypically ‘female’ medical disorders such as chronic fatigue syndrome/myalgic encephalomyelitis, anorexia nervosa and migraine.FND is a common and disabling disorder presenting with a variety of clinical manifestations, including weakness, sensory changes, involuntary movements, gait disturbance, dissociative episodes and speech problems. It is described as a multinetwork disorder involving abnormalities within and across brain circuits implicated in emotion processing, attention, interoception, speech, motor control and sense of agency, among other functions.Despite this, people with FND seeking treatment often wait years to receive treatment, with some patients waiting a mean time of 8 years before the diagnosis is made.Much has been written about the history of FND, some of which includes themes of maltreatment of women.We, a group of clinicians and academics who research and treat patients with FND, recognise that many people do feel ignored or dismissed on receipt of an FND diagnosis. Some perceive that the diagnosis of FND has been used euphemistically to denote an illness that is imagined, or worse, feigned.We are a group of women clinicians and academics, and we believe that people of all gender identities, and the conditions which affect them, should be equally respected and have parity of access to funds for investigation and treatment. Building on a core definition of feminism as the belief that women should be afforded the same rights, power and opportunities as men and be treated in an equitable way, we further note clinical ethicist Professor Wendy Roger’s view that “… a feminist approach to health inequities leads us to examine the connections between disadvantage and health, and the distribution of power in the processes”.Stigma is pervasive in FND and occurs in many forms, subtle and overt. The origins of this stigma are complex and may arise from issues around FND’s associations with psychiatric diagnoses and misperceptions of the degree of voluntary control held by patients. This mirrors the stigma seen in other functional disorders such as fibromyalgia, and psychiatric diagnoses such as emotionally unstable personality disorder, and indeed in depression and anxiety disorders—all diagnoses that are more prevalent in women than men.Undoubtedly, progress has recently been made. However, people with FND continue to describe experiences of doubt, blame and of being seen as less ‘genuine’ than those with other disorders, particularly ones with more obvious structural pathology.Lack of undergraduate and postgraduate education in FND means that well-intentioned junior clinicians are vulnerable to the repeated hits of the ‘hidden curriculum’.These old-fashioned but ever present and stigmatising attitudes are further fuelled by misunderstanding around the aetiology and presentation of FND. In our experience, clinicians, patients and caregivers raise the following tropes about FND that can perpetuate stigma: (1) FND represents a missed alternative diagnosis; and (2) FND is thought of as arising from a disembodied ‘mind’ in an extreme dualistic model of the mind as separate from the body.To address the first point, although we cannot ignore occasional misuse of the term, FND diagnoses when made appropriately are stable, reliable and unlikely to represent a missed alternative diagnosis. Descriptions of clinical presentations of the disorder we presently call FND are remarkably stable geographically and historically.Diagnoses of FND, just like those of migraine, Parkinson’s disease or motor neuron disease, are primarily made in the clinic, on the basis of reproducible history or signs, before imaging or other investigations are undertaken. Signs which have been proven to be reliable in motor FND include Hoover’s sign, hip abductor sign, drift without pronation, identification of typical FND gait, and ipsilateral weakness of the sternocleidomastoid with hemiparesis.Physicians seem to fear giving an incorrect diagnosis. But in a review of 27 studies with FND, with a total population of 1466, the proportion of misdiagnosis was less than 4% after an average of 5 years of follow-up.To address the second point, that FND lacks validity on the basis that symptoms are feigned or imagined, there is simply little evidence of this. The persistence of positive signs used to identify FND such as contractures in patients with fixed dystonia, persistence of Hoover’s sign even after the patient has been shown how it operates, evidence of shoe wear in patients with functional gait disorders, stability in presentation and improvement with therapy are some of the many features that indicate FND is not compatible with feigning.Furthermore, functional and structural changes in the brains of people with FND have demonstrated evidence of increased connectivity between motor control and emotional processing areas.This brings us to our third point; FND cannot be assigned to a simple psychological or physical category. Simply put, our brain is a complex organ, responsible for emotions, attention, movement, sensations and predictions about the world. All of these aspects exert influence on each other and are shaped by our experiences—be it life events, neurological illness or something else we cannot yet identify. It is impossible to disentangle these aspects of functioning from each other. Dated ‘conversion’ theories, that trauma is always the underlying cause of functional symptoms, are too simplistic, often do not make sense to patients and have rightly been removed as essential criteria from international classifications.This is not to undermine the importance of life events in the aetiology of FND. A recent systematic review and meta-analysis found maltreatment was substantially more common in people with FND than in healthy controls and patient controls.The Handmaid’s TaleWe lived in the gaps between the stories—Margaret Atwood, FND is a feminist issue. We say this because (1) FND predominantly affects women; (2) historical and societal issues affecting women continue to shape the narrative of FND; (3) under-recognition of FND occurs in men due to potential diagnostic bias; (4) sexual abuse and violence are gender-weighted risk factors for FND; (5) socioeconomic disparity exists between men and women, contributing to inequalities in access to treatment; (6) FND clinical services and research are chronically underfunded, in line with the neglect of disorders disproportionately affecting women.Functional disorders, including FND, have a problematic history. These conditions, among other disorders originating from the brain, such as epilepsy and psychotic disorders, were historically drowned in prejudice and even punishment; and what would be described today as FND has been depicted in terms of moral failing, demonic possession, hysteria or witchcraft; with uterine repositioning reported to be a proposed treatment.These accounts of treatment, while extreme, unfortunately parallel accounts given by patients with FND today. We continue to hear of patients with FND being ‘shamed, blamed and humiliated’ on account of their diagnosis.The prejudicial treatment of women with functional disorders is evident in a recent longitudinal study by Ballering and colleagues, describing the management of persistent somatic symptoms, most of which were likely functional in origin. They showed that women presenting with symptoms of dizziness, tiredness, pain and tingling were less likely to receive a physical examination, diagnostic imaging and specialist referral for their complaints than men. They were also less likely to receive a clear diagnosis for their symptoms.However, historical biases may make physicians more likely to diagnose FND in women than in men, despite similar symptom profiles.Yet against this backdrop of changing terminology and prejudice, careful analysis of historical case records tells us that clinical presentations of FND (in both women and men) have in fact remained remarkably consistent over the last century.Briquet, a French physician, was one of the first to describe and support the diagnosis of hysteria in men, although he did not agree with the use of the term hysteria.10et alThere is a large amount of data suggesting that FND affects more women than men.et al found that male gender significantly impacted diagnostic agreement in functional movement disorders.Future research is needed to explore the reasons for these differences—with attention to biological, psychological and social factors. While we are not disputing that FND is more common in women, as the evidence clearly demonstrates, there may be some limitations to these epidemiological figures. It is possible there may be some physician bias against diagnosing FND in men, given the way FND has been (and continues to be) portrayed and taught. Morgante The elevated rates of FND in women might reflect complex differential exposures to social and environmental risk factors. Women are far more likely to have experienced childhood sexual abuse, intimate partner violence and sexual assault than men.et al found that life events—most commonly maltreatment in the form of neglect, physical and sexual abuse—were experienced by patients with FND eight times as frequently as healthy controls and twice as often as in other psychiatric and neurological conditions.et al expanded on the review, finding that family, relationship and work adverse events were also more common in patients with FND than controls—where the control group were patients diagnosed with another psychiatric or neurological condition.A recent important study found a significant association between sexual abuse and FND in women, and a greater likelihood that women who are sexually abused will develop functional movement disorders than men who are sexually abused.Associations between women, violence and FND span cultures and ethnicities. A large Bolivian study showed that rates of functional seizures were much higher in women who suffered psychological, sexual and physical intimate partner violence than those who did not.There are issues that, worldwide, disproportionately affect women. Estimates published by WHO indicate that globally almost 1 in 3 (30%) of women worldwide have been subjected to either physical and/or sexual intimate partner violence or non-partner sexual violence in their lifetime.et al.There may be additional, more ‘hidden’ gender-weighted risk factors outside trauma that are important in the aetiology of FND, such as societal and familial expectations. This may be partly captured in the ‘family events’ noted in the above review by Morsy Although data are lacking, we also acknowledge the likely intersection of other biases and inequalities as contributors to stigma and discrimination experienced by transgender women and non-binary people with FND.Women’s health status is significantly lower than men’s across the globe, and this disparity is associated with education, employment and economic status.Access to appropriate diagnostics and treatments for FND remains largely dependent on socioeconomic status, with a clear relationship demonstrated between income and access to specialist expertise for patients with FND.The situation around allocation of resources to FND research parallels that of females in research generally. Females are considerably under-represented in research. Reasons for the exclusion of female subjects in medical trials include hormonal differences, cost and lack of comparability with previous trials in solely male participants.In an examination of allocation of funding to medical research, Mirin outlines striking disparities in funding relative to disease burden between diseases that primarily affect men, and those that primarily affect women—chronic fatigue syndrome/myalgic encephalomyelitis, migraine and other headache disorders being top of the list.Nothing I accept about myself can be used against me to diminish me.—Audre Lorde, Sister OutsiderThe history of FND, in some ways, mirrors the history of women in society. It is a history laden with inequalities, dismissal and injustice which cannot be undone. Now, patients with FND do not need pity, but parity. The inequalities and injustices continue today in the form of ignorance of FND in teaching curricula, delayed diagnosis, stigmatising healthcare interactions, paucity of specialist services and an underfunding of scientific research. But discrimination is not an inevitable component to the FND diagnosis, which, when it is properly made, is as stable and accurate as that of any other common neurological disorder.We are not the first group to highlight that FND critically requires parity of esteem with neurological conditions of equivalent epidemiological and economic importance.This is a call to action. We support and urge careful and appropriate use of this diagnosis to support and empower those affected by FND so their symptoms and suffering can be recognised and validated. We call for respectful models of clinical care and an end to dismissive and harmful language and behaviour towards people with FND. We call for a shift in approach to FND, and other functional disorders, to move away from dualist models of mind and body. We call for parity for FND among other neurological disorders in medical education, and better training curricula for all the allied professions that have contact with patients with FND.We call for more recognition and funding for more impactful laboratory and clinical research, and support of female leadership in the FND community to improve diversity and excellence in the field. We urge planning and funding for better and more universally available FND treatment services, and a universal upskilling of clinicians, so that people with FND can finally receive the treatment they require and deserve.


Here, we report a case of left apicoposterior (S In anatomical lung resection in patients with incomplete interlobar fissure, dissection of the interlobar parenchyma can cause hemorrhage and air leaks. Fissureless techniques, which involve dissecting the lung parenchyma using mechanical staplers at the final step after dividing hilar bronchovascular structures, are used in lobectomy to avoid dissecting the interlobar parenchyma . HoweverNear-infrared (NIR) thoracoscopy with indocyanine green (ICG) is used for intersegmental plane identification in segmentectomy . This te1+2) segmentectomy using NIR thoracoscopy with ICG to identify the separation range of interlobar fissure after dissecting the relevant vessels.We report a case of left apicoposterior (S1+2)  in the left apicoposterior segments  and the apicoposterior artery branches (A1+2a+b and A1+2c) were dissected clockwise from the ventral side to the dorsal side of the pulmonary hilum. After intravenous ICG (0.3mg/kg/10ml) bolus administration, NIR thoracoscopy  identified the intersegmental plane and the separation range of interlobar fissure  was dissected, and finally, the intersegmental plane was created using mechanical staplers, completing the left S1+2 segmentectomy. No intraoperative air leaks occurred. The patient experienced no complications and was discharged 10 days after surgery.Video-assisted thoracoscopic surgery left Se Figure . The apie Figure . The lime Figure . FollowiNIR thoracoscopy with ICG in thoracic surgery is a relatively new technique, initially used for intersegmental plane identification in segmentectomy  and receThis method has the advantage of minimizing bleeding and air leaks while still allowing for essential and sufficient interlobar dissection. It has been reported that the frequency of pulmonary fistulas and the local relapse rate in segmentectomy are higher than in lobectomy . This apThe key aspect of this method is the preliminary dissection of relevant pulmonary arteriovenous vessels. Administering ICG after dissecting the relevant vessels allows identification of the intersegmental plane and the separation range of interlobar fissure. On the other hand, this method is unsuitable for cases where dissection of the relevant vessels is difficult to perform. NIR thoracoscopy with a transbronchial injection of ICG has been reported  and may Finally, we would like to mention two points of caution regarding the use of NIR thoracoscopy with ICG intravenous injection. The first point is that administration is contraindicated in cases with a known allergy to the contrast agent. Moreover, while ICG is generally regarded as safe, serious adverse reactions have been recorded in 0.05% of cases . The sec1+2 segmentectomy with incomplete interlobar fissure, by dissecting relevant vessels first, the separation range of interlobar fissure was easily identified using NIR thoracoscopy with ICG. This technique is considered to be useful in reducing the risk of hemorrhage and air leaks.In S