Mother-to-child transmission (MTCT) of human T-cell leukemia virus type 1 (HTLV-1) is a major cause of adult T-cell leukemia (ATL). Owing to the poor prognosis of ATL and the fact that more than one million people have been infected with this virus, the HTLV-1 antibody screening test was established in Japan in 2010 for all pregnant women to detect carriers and prevent MTCT. Because breastfeeding is the most common route of postnatal MTCT, exclusive formula feeding is widely used as a measure to prevent MTCT. Recently, it was reported that there is no obvious difference in the efficacy of short-term breastfeeding for ≤3 months in preventing MTCT compared to that in exclusive formula feeding, and that a duration of breastfeeding that does not exceed four months can be effective for preventing MTCT.Approximately 95% of mother-to-child transmission (MTCT) of human T-cell leukemia virus type-1 (HTLV-1) is derived from prolonged breastfeeding, which is a major cause of adult T-cell leukemia (ATL). Exclusive formula feeding (ExFF) is therefore generally used to prevent MTCT. A recent cohort study revealed that 55% of pregnant carriers chose short-term breastfeeding for ≤3 months in Japan. Our meta-analysis showed that there was no significant increase in the risk of MTCT when breastfeeding was carried out for ≤3 months compared with ExFF (pooled relative risk (RR), 0.72; 95% confidence interval (CI), 0.30–1.77), but there was an almost threefold increase in risk when breastfeeding was carried out for up to 6 months . Thus, short-term breastfeeding for ≤3 months may be useful in preventing MTCT. Breastmilk is the best nutritional source for infants, and any approach to minimizing MTCT by avoiding or limiting breastfeeding must be balanced against the impact on the child’s health and mother–child bonding. To minimize the need for nutritional interventions, it is necessary to identify factors that predispose children born to carrier mothers to MTCT and thereby predict MTCT development with a high degree of accuracy. Early-life exposure to infectious agents may be involved in the development of future cancers. Well-known pathogens include the human papillomavirus, hepatitis B and C viruses, Epstein–Barr virus, and human T-cell leukemia/lymphoma virus type-1 (HTLV-1) . While tA recent meta-analysis demonstrated that the risk of all-cause death was higher in people with HTLV-1 infection than that in people without the infection . The anaDiverse clinical features, including lymphadenopathy, skin lesions, increased abnormal lymphocytes, frequent blood and bone marrow involvement, hypercalcemia, and lytic bone lesions characterize ATL . The diaATL has been associated with HTLV-1 mother-to-child transmission (MTCT) ; (2) theIn Japan, there has been a nationwide antenatal HTLV-1 antibody screening program since 2010 to prevent HTLV-1 MTCT ,25. BecaHowever, despite the implementation of nationwide antenatal HTLV-1 antibody screening, there is no consensus among healthcare providers, especially regarding prevention by nutritional regimens. In this review, the mechanisms of MTCT and evidence for preventive measures through nutrition will be discussed along with the latest findings.The implementation of nationwide HTLV-1 antibody screening tests in all pregnant women is controversial. Although the United Kingdom National Screening Committee considered the antenatal HTLV-1 screening program three times, the committee did not recommend introducing a screening program in the United Kingdom because of the low prevalence of HTLV-1 infection and the low risk for infected infants developing serious illness . AlthougThe flowchart for HTLV-1 carrier screening during pregnancy in Japan is shown in The prevalence of HTLV-1 carriers among pregnant Japanese women in 2011 and 2013 was 0.15% and 0.18%, respectively . In 2019Based on available data in 2012, Gessain and Cassar reported that the most endemic regions for HTLV-1 are the Southwestern part of Japan, sub-Saharan Africa and South America, the Caribbean region, and foci in the Middle East and Australo-Melanesia. They also reported the prevalence of HTLV-1 in pregnant women in these regions . RosadasHuman immunodeficiency virus (HIV), mouse mammary tumor virus (MMTV), and HTLV-1 are transmitted from mother to child through breast milk. HIV MTCT can occur before, during, and after delivery, with postnatal transmission through breastfeeding accounting for one-third to one-half of all cases of HIV MTCT . Both ceIn contrast to HIV and MMTV, HTLV-1 cell-free viruses are rarely detected extracellularly ,44. Cell+ T cells via their cellular receptors such as heparin sulfate (HS) proteoglycans and neuropilin 1 (NRP-1), which are used for the initial binding to the cell, and glucose transporter 1 (GLUT1) for entry . T. Tp = 0.In our cohort study and meta-analysis, there was no obvious difference in the MTCM rate between ExFF and STBF ≤ 3 months. In addition, data on FTBMF are lacking. Therefore, from the perspective of preventive effects alone, either ExFF or STBF may be acceptable for postnatal MTCT prevention. However, there are some considerations and issues that need to be addressed when choosing between these nutritional approaches .It is less challenging to perform ExFF than it is to perform STBF, but a major drawback of ExFF is that it does not offer the benefits of breastfeeding. In addition, there are issues regarding the risk of postpartum depression due to low self-esteem owing to not being able to breastfeed and anxiety about the development of HTLV-1-associated diseases . TherefoSTBF for ≤3 months appears to offer the advantages of breastfeeding over ExFF, in that the carrier mother can feed her own milk even if it is only for a few months. However, it may not always be easy to refrain from breastfeeding after 3 months, as evidenced by the results of our cohort study . This maFTBMF is very time-consuming when performed at home. Even in our cohort study, only 6.1% of carrier pregnant women opted for it . Given tRegardless of its duration, breastfeeding may also be combined with the use of infant formulas. In recent studies, the rate of MTCT of HIV was extremely high, at approximately 20%, compared with normal breastfeeding or infant formula feeding . It is sHuman milk donated to breast milk banks should be screened for maternal HTLV-1 infection . In theoThe selection of a feeding regimen is an important factor associated with MTCT of HTLV-1, but the involvement of other factors should also be considered.Plancoulaine et al. conducted a large genetic epidemiological survey in an HTLV-1-endemic population of African origin from French Guiana. They found the presence of a dominant major gene that predisposes to HTLV-I infection, in addition to the expected familial correlations (mother–offspring and spouse–spouse) due to the virus transmission routes. The authors concluded that this gene appears to account for most infections occurring in children through breastfeeding and can explain, at least in part, the reason why MTCT of HTLV-I only occurs in a certain proportion of children fed by infected mothers ,119. A fAn immunological issue that has long been of interest is whether transfer antibodies or antibodies contained in breastmilk play an effective role in preventing mother-to-child infection. Pregnant women infected with HTLV-1 have significantly increased levels of anti-HTLV-1 antibodies, although PVL does not change during pregnancy . This isSubstances contained in breastmilk, such as tumor growth factor-β and lactoferrin, which are rich in colostrum ,126, proSince the discovery of HTLV-1 almost 40 years ago, much has been learned about the associated disease and its pathogenesis. MTCT of HTLV-1 became evident within a short time after its discovery, and epidemiological studies and animal studies have shown that prolonged breastfeeding is an important risk factor for MTCT. However, symptoms are rarer in infected infants and children than in adults, and for this reason, there have been no vigorous studies of MTCT, and the detailed mechanisms underlying MTCT remain unknown. Our recent cohort studies and meta-analyses have shown that STBF ≤ 3 months is not significantly different from ExFF in preventing MTCT, which may provide reassurances that STBF can be successfully implemented. However, STBF and ExFF may not be optimal interventions for carrier pregnant women and their children. Breastmilk is the best source of nutrition for infants, and any approach to preventing MTCT by avoiding or limiting breastfeeding must be balanced against the impact on the child’s health and mother–child bonding. In the absence of commercially available vaccines, antivirals, and neutralizing antibodies, to minimize the need for nutritional interventions, it is necessary to identify factors that predispose children born to carrier mothers to MTCT and to thereby predict the development of MTCT with a high degree of accuracy. In addition, further research is needed on the mechanisms underlying prenatal MTCT and its prevention.


Fertility is fundamental to reproductive success, but not all copulation attempts result in a fertilized embryo. Fertilization failure is especially costly for females, but we still lack a clear understanding of the causes of variation in female fertility across taxa. Birds make a useful model system for fertility research, partly because their large eggs are easily studied outside of the female's body, but also because of the wealth of data available on the reproductive productivity of commercial birds. Here, we review the factors contributing to female infertility in birds, providing evidence that female fertility traits are understudied relative to male fertility traits, and that avian fertility research has been dominated by studies focused on Galliformes and captive (relative to wild) populations. We then discuss the key stages of the female reproductive cycle where fertility may be compromised, and make recommendations for future research. We particularly emphasize that studies must differentiate between infertility and embryo mortality as causes of hatching failure, and that non-breeding individuals should be monitored more routinely where possible. This review lays the groundwork for developing a clearer understanding of the causes of female infertility, with important consequences for multiple fields including reproductive science, conservation and commercial breeding. DespiSuccessful fertilization occurs when a male pronucleus and a female pronucleus fuse to form a zygote (i.e. syngamy) . We therGallus gallus domesticus) and turkey , hatching failure is still a pervasive issue in many commercial breeds, and the reasons for this are not fully understood . M. MNymphilchicus) . Inflamm. 3.1.3 3.1.3.1A wealth of experimental evidence in birds (mostly poultry) shows that diet strongly influences egg production and fertility. Striking the balance between optimum nutrient uptake (to maximize production) and nutrient toxicity and/or obesity is of primary concern to the poultry industry . Modern 12, protein and linoleic acid .,28.17,28just one ,207. Whejust one ,206.Supernumerary male pronuclei are probably degraded by DNAses in the germinal disc and PVL of mature oocytes , though . 4It is clear that females can exert far more control over fertilization than has historically been assumed, but if and how females influence whether their ova are successfully fertilized is often ignored in favour of male processes  . Here weWhen investigating reproductive failure, the fact that infertility and embryo mortality are fundamentally distinct processes needs to be explicitly acknowledged. Specifically, that infertility is used to describe failed fertilization, and any process contributing to failed fertilization is a mechanism of infertility . Similarly, if an egg fails to hatch but was fertilized, then the cause of hatching failure must be referred to as embryo mortality, even if development arrested after only a few cell divisions. If fertility status cannot be unequivocally determined using the appropriate techniques , then thThe female reproductive tract typically offers a hostile environment for sperm, providing considerable potential for female processes to influence sperm survival and transport to the ovum. While the processes of sperm selection, storage, release and transport within the reproductive tract have received increasing research attention over the past few decades, we still lack fundamental understanding of the underlying mechanisms, and the degree of intra- and inter-specific variation in these processes, with the vast majority of work having focused on a very limited number of domestic species. Many of the female-mediated processes required for high fertility also deteriorate to some degree with age, making fertility problems more likely in older birds. This may have particularly important consequences for captive and managed threatened populations, where individuals may reproduce to an older age than their wild counterparts, due to reduced predation and competition pressure, and high accessibility of food and other resources. The field of avian reproductive science will also benefit from better understanding the impact of other factors on female fertility, such as stress, hormonal and physiological disorders (particularly in wild birds where less is known), environmental pollutants, intra- and inter-individual variation in egg production, egg quality, sperm selection and the female immune response within the oviduct (including the ovaries).The causes and maintenance of variation in fertility is a key question in evolutionary biology, and one in which the role of the female is often sidelined. Our hope is that this review challenges the field of avian reproductive science and evolutionary biology to consider female processes to a greater degree when investigating the causes of depressed fertility in birds. Using birds as a model system for the study of female fertility across taxa presents several advantages and will provide insights not only in the field of reproductive biology, but also for fields such as conservation and commercial animal breeding as well.Click here for additional data file.


Editor,Acute uric acid nephropathy is one of the causes of acute kidney injury (AKI), which usually occurs in patients with tumor lysis syndrome and postoperative cardiopulmonary , 2. AKI 2 microglobulin increased mildly. Hepatitis B virus (HBV) surface antigen, HBV e-antigen and HBV core antibody were positive in the serum. Hemodialysis was used to remove metabolic wastes. Febuxostat, methylprednisolone and tenofovir alafenamide fumarate were also added. One day after treatment, levels of serum UA, Scr and BUN quickly corrected to 671 μmol/L, 537 μmol/L and 17.26 mmol/L significantly with clinical symptoms resolved. Maintenance therapy with hemodialysis was used again on day 3. After 5 days’ treatment, levels of serum UA, Scr and BUN were decreased to 461 μmol/L, 163 μmol/L and 9.76 mmol/L, respectively, with sustained urine volume being 1500–2500 mL. On day 7, he was discharged with the levels of Scr and BUN normal  and serum UA being 440 μmol/L.A 27-year-old male with a history of gout diagnosed 2 years ago was admitted for nausea, vomit and serum creatinine (Scr) increasing for 1 day with no significant past history and medical history. His eating habits were profound intake of purine-rich foods. Physical examination showed: temperature was 37.4 °C, blood pressure was 140/93 mmHg, ankle edema, and no other abnormalities. Investigations revealed extremely elevated UA, Scr and blood urea nitrogen (BUN), which increased to 1123, 676 and 23.54, respectively, from reference values  and decreased UA in the urine of 1187.5 (reference range was 2400–5400 μmol/24 h). The urine UA-to-creatinine ratio was 1.76 and a large number of uric acid crystals were found in the urinary sediment, which was consistent with acute uric acid nephropathy [AKI can be caused by hyperuricemia alone. A high-purine diet, increased purine metabolism and excessive alcohol consumption could result in increasing UA production rapidly . Uric acIn conclusion, this was a unique case of AKI caused by extremely high increase in serum UA level alone. The pathologic features of hyperuricemia-induced AKI are reversible. With timely recognition and management, the renal function can be restored completely.


Interestingly, MAI was more reduced in RBD+P compared to iRBD patients. In comparison, CCC was higher in iRBD patients compared to controls whereas CCC was lower in RBD+P groups compared to control and iRBD groups in various frequency bands during both NREM N2 and REM sleep stages. Among them, increased CMC during REM sleep revealed correlation between clinical severities of RBD symptoms. Our findings indicate that MAI, CMC, and CCC showed distinctive features in iRBD and RBD+P patients compared to controls, suggesting potential usefulness to understand possible links between these diseases.REM sleep behavior disorder (RBD) could be a predictor of Parkinsonism even before development of typical motor symptoms. This study aims to characterize clinical features and corticomuscular and corticocortical coherence  during sleep in RBD patients with or without Parkinsonism. We enrolled a total of 105 subjects, including 20 controls, 54 iRBD, and 31 RBD+P patients, patients who were diagnosed as idiopathic RBD (iRBD) and RBD with Parkinsonism (RBD+P) in our neurology department. We analyzed muscle atonia index (MAI) and CMC between EEG and chin/limb muscle electromyography (EMG) and CCC during different sleep stages. Although differences in the CMC of iRBD group were observed only during REM sleep, MAI differences between groups were noted during both REM and NREM N2 stage sleep. During REM sleep, CMC was higher and MAI was reduced in iRBD patients compared to controls ( The third edition of the International Classification of Sleep Disorders ICSD-3) updated the diagnostic criteria for REM sleep behavior disorder (RBD), by repeated episodes of sleep-related vocalization and/or complex motor, and often violent, dream enactment behaviors, based on polysomnography (PSG) by demonstrating REM sleep without atonia (RWA) ,2,3. RBD updated Although idiopathic RBD (iRBD) patients do not show Parkinsonian motor symptoms, they sometimes present non-motor symptoms similar to PD ,12. PD pOn the other hand, functional connectivity using various quantitative physiologic markers has been investigated to study physiologic and/or pathologic neural activities. Among various physiological markers, corticocortical coherence (CCC) and corticomuscular coherence (CMC) have been applied to investigate functional connectivity by measuring synchronized oscillatory activities between different cortical regions , or betwThis study aimed to characterize the clinical and polysomnographic findings as well as electrophysiologic markers in RBD patients with or without Parkinsonism. To investigate the differences of corticocortical and corticomuscular connections including locomotor drive across stages of sleep, CCC and CMC, in addition to MAI were compared in control and RBD patients with or without Parkinsonism. We chose these indices because synchronization between different brain regions as well as corticomuscular connections could be involved as common pathophysiologic changes both in RBD and Parkinsonism patients, which would help us understand possible links between these diseases and underlying mechanisms of phenoconversion of iRBD to neurodegeneration.All patients were recruited had visited outpatient sleep clinic at Ewha Womans University Mokdong hospital and been diagnosed with RBD between 2013 and 2019. Since RBD might be considered as one of the cardinal symptoms of neurodegeneration, especially for PD, we evaluated RBD patients on whether they had Parkinsonian symptoms or not. Specifically, we checked these patients for Parkinsonian motor symptoms such as bradykinesia, rigidity, tremor at rest, as well as postural instability, which was evaluated by a neurologist using Unified Parkinson’s disease scale part 3 (UPDRS-III) . SometimSeventy-six RBD patients were identified. The diagnosis was based on the ICSD-3 criteria, which includes the existence of dream enacting motor activity associated with RWA, the presence of excessive tonic or phasic chin electromyographic (EMG) activity during REM sleep. Among them, 54 patients comprised the iRBD group and the remaining 22 were the RBD combined with Parkinsonism (RBD+P) group including 18 PD and 4 DLB patients. The disease duration was 6.9 years on average from the clinical diagnosis. As Control group, we recruited 20 age-matched healthy subjects who did not have a history of RBD or other chronic neuropsychiatric diseases.We excluded subjects who had history of head trauma, encephalopathy, cerebrovascular disease, or severe diabetes mellitus; who were taking medication known to affect REM sleep including antidepressants and sedatives; or who had cognitive dysfunction with a Korean version of Mini-Mental Status (K-MMSE) score equal to or lower than 24; or who had respiratory disturbance index (RDI) higher than > 15/hour based on PSG, similar to the previous study . Thus, aAll evaluations were performed by a neurologist. All patients had completed the Korean version of REM sleep behavior disorder questionnaire (RBDQ-KR) for evaluation of clinical severity, based on previous literature ,31,32,33®PSG Clinical Software . The video-PSG recording including 10–20 system electroencephalography (EEG), chin EMG, electrocardiography, snoring assessment, pulse oximetry, and airflow assessment was performed during one full-overnight monitoring in the sleep laboratory. Sleep stages were scored by a sleep specialist and experienced polysomnographers according to standard criteria of American Association Sleep Medicine (AASM) manual [All participants attended a video-PSG recording using a comprehensive sleep laboratory with a Twin) manual . RBD wasThe PSG characteristics of RBD are characterized by either or both the following features: (1) Sustained muscle activity in REM sleep in the chin EMG or (2) excessive transient muscle activity during REM in the chin or limb EMG. Sustained muscle activity (tonic activity) in REM sleep was defined as an epoch of REM sleep with at least 50% of the duration of the epoch having a chin EMG amplitude greater than minimum amplitude than in NREM. Excessive transient muscle activity (phasic activity) in REM sleep defined when in a 30-s epoch of REM sleep divided into 10 sequential 3 s mini-epochs, at least 5 (50%) of the mini-epochs contain bursts of transient muscle activity. In RBD, excessive transient muscle activity bursts are 0.1–5.0 s in duration and at least 4 times as high in amplitude as the background EMG activity ,30,34,37In previous literature, the atonia index and CMC were significantly different between control and iRBD groups only during REM sleep ,33. A moIn addition to the standard PSG, we recorded scalp EEG from 19 electrodes according to the international 10–20 system during the full-night sleep study. Similar to the previous studies ,33, we uWe evaluated muscle atonia index (MAI), beta-band CMC between scalp EEG and chin/limb EMG, and CCC between intrahemispheric/interhemispheric electrodes. Ferri’s method was used to calculate MAI ,19. The http://www.neurospec.org/) (accessed on 23 Mar 2019) was used to calculate the coherence after EMG and EEG signals were rectified in 30-s epochs to enhance the firing rate of the signals [In order to evaluate CMC, coherence was computed between four pairs of chin/limb EMG signals and central region EEG signals: chin EMG-C3 EEG, chin EMG-C4 EEG, right limb EMG-C3 EEG, and left limb EMG-C4 EEG. The Neurospec 2.0 Matlab toolbox  of Ewha Womans University School of Medicine approved this study. Requirement for informed consent was waived for this retrospective analysis of clinical data.p < 0.05.Descriptive data are presented as mean ± standard deviation or frequency (percentage). Group comparisons of demographic, clinical, and PSG findings; CMC; MAI; and CCC among control, iRBD, and RBD+P groups were performed. Analysis of variance (ANOVA) or Kruskal–Wallis was used for evaluation of continuous variables and dichotomous variables, and post hoc significance was assessed with the Tukey–Kramer test using SPSS 20.0  with the statistical significance defined as Overall, we enrolled 20 healthy controls, 54 patients for iRBD, and 22 patients for RBD+P group (18 PD and 4 DLB). Their clinical characteristics are summarized in In PSG findings, the numbers of patients with N3 stage NREM sleep in iRBD and RBD+P groups was reduced compared to that in the control group. PLM score was higher in both iRBD and RBD+P groups compared with the control group. The PSG findings are summarized in The CMC was evaluated in each stage and is summarized in The MAI was significantly decreased during both NREM2 and REM sleeps in the RBD+P group compared with iRBD group . The iRBThe degree of coherence between intrahemispheric/interhemispheric electrodes is believed to indicate the power of the interconnections. Generally, it has been considered that theta and alpha bands reflect resting state activities during sleepiness and/or waking states, beta band for sensorimotor activities, whereas gamma band for higher cortical, often cognitive activities. During NREM N2 sleep stage, the iRBD group revealed a lower alpha (8–12 Hz) and gamma (30–55 Hz) band coherence in frontal, temporal, and parietal areas compared with control group, whereas higher alpha band coherence in frontal, central, and temporal areas, higher beta (13–30 Hz) band coherence in parietal and occipital areas, compared with RBD+P patients. In RBD+P patients, CCC in frontal, central, and temporal alpha power, and CCC in frontal, central, temporal gamma power during NREM2 sleep were lower than controls, and also CCC in frontal, central, and temporal alpha power, CCC in central, parietal, and occipital beta power, as well as CCC in frontal, central, temporal, and occipital gamma power were lower than the iRBD group A.During REM sleep, iRBD patients showed a considerably higher CCC in theta and alpha bands in frontal, central, and occipital areas compared with control subjects. The RBD+P group showed a lower CCC in beta band in frontal and temporal areas, and lower CCC in gamma band in frontal, temporal, parietal, and occipital areas than the control group. In addition, the RBD+P patients had a lower delta (2–4 Hz), theta (4–7 Hz), alpha, beta, and gamma band CCC in central, temporal, parietal, and occipital areas compared with iRBD patients B.This study characterized clinical and polysomnographic findings in RBD patients with or without Parkinsonism. We also investigated the differences of muscular and cortical activities by determining MAI, CMC, and CCC during NREM N2 and REM sleeps in control and RBD patients with or without Parkinsonism. The main findings of this study were: (i) that the MAI was reduced in NREM N2 and REM sleeps in iRBD compared to controls, but more reduced in RBD+P compared to controls and iRBD groups. (ii) The CMC was significantly increased during REM sleep in iRBD compared to controls, and tended to be increased during REM sleep in RBD+P without statistical significance. (iii) CCC in theta and alpha frequency bands in frontal, central, and occipital areas were increased in iRBD compared to controls, and higher theta and alpha CCC in central, parietal, and temporal areas in iRBD compared to RBD+P group, and lower CCC in various frequency bands in RBD+P compared to controls and iRBD groups. Interestingly, higher CMC values during REM sleep were correlated with more severe RBD symptoms based on RBDQ-KR in both iRBD and RBD+P groups.We observed clinical differences between iRBD and RBD+P groups in systolic BP drop and orthostatic abnormalities. Compared to the control group, the RBD+P group demonstrated significant olfactory dysfunction. A research group conducting a small case-control study previously reported on abnormalities of motor, olfaction, and autonomic function in iRBD patients .PSG findings demonstrated that overall sleep quality did not show major differences among groups except for decreased amount of N3 sleep in iRBD and RBD+P groups. However, both iRBD and RBD+P groups showed higher PLM scores compared with the control group. The correlation between RBD and PLM has been identified in iRBD patients , and theThe CMC values showed significant differences between iRBD and control groups during REM sleep, but there were no differences among groups during NREM N2 sleep. The MAIs were distinct among groups during both NREM N2 and REM sleep stages, especially between iRBD and RBD+P groups. In previous studies, the iRBD group had higher beta frequency CMC and lower MAI than controls during REM sleep . Beta-baDuring REM sleep, the CCC values between iRBD and control groups documented significant differences in frontal and occipital areas. While differences between iRBD and RBD+P groups during NREM N2 sleep were recorded in faster frequency bands, differences between iRBD and RBD+P groups during REM sleep was prominent in slower frequency bands, mainly in motor and supplementary motor areas and in part of the premotor area. Previous study of EEG in RBD determined that RBD patients had considerable occipital frequency during REM sleep in spite of prominent beta power . ConcomiTo examine whether these indices could reflect clinical severity of the RBD symptoms, we examined the relationship between these values and scores of RBDQ-KR. Although the RBDQ is based on a subjective questionnaire, a score of RBD symptoms from patients and/or caregivers, it is commonly used as one of the standard diagnostic instrument , especiaThe methodological issues should be also noted. We used rectified EEG signals to calculate CMC and CCC, based on the previous literature, where time-frequency analysis was also used to compare the differences between iRBD and control groups . In the The sleep architecture of patients did not show major difference compared to controls, except significantly decreased NREM N3 and non-significant tendency of increased NREM N1 in iRBD and RBD+P groups compared to controls. Overall percentages of NREM N1 seem to be slightly increased and N3 to be decreased in our subjects compared to the reference data from previous literatures ,56. WASOIn summary, this study characterized clinical and polysomnographic characteristics of controls and patients with idiopathic RBD and RBD+P. We also investigated differences in locomotor driving and corticocortical coherence in controls, iRBD patients, and RBD+P patients. These findings showed clear distinctions between healthy control, iRBD, and RBD+P groups. The CMC values documented differences between controls and iRBD patients. In particular, the MAI and/or CCC values were more prominently different between iRBD and RBD+P groups, suggesting the possibility of development of neurodegenerative disorders in those with newly diagnosed RBD.The limitations of this study may include the inability to detect developments or changes in the characteristics of the target population among the groups because the comparisons were performed in a cross-sectional study setting, not a longitudinal design. We only used subjective score for evaluation of RBD clinical severity, and the PSG data from only one sleep center. There is another possibility of resemblance of PD and other diagnoses such as atypical Parkinsonian disorders including PSP considering that it is difficult to differentiate PD from and PSP in early stages ,58. ThisAlthough it was not a longitudinal study setting, the current study showed that CMC and MAI were different in iRBD and RBD+P groups compared to controls, which was somewhat confirmatory to the previous literatures. In addition, we found that the clinical severity of RBD revealed significant correlation with higher CMC values during REM sleep in both iRBD and RBD+P groups. However, CMC and RBDQ-KR are indirect and subjective parameters, and correlation between CMC and RBDQ-KR at cross-sectional time points could not directly apply to the prediction of the disease progression, for instance, the future development of PD in RBD patients. Although the MAI could be a more plausible index, the lack of correlation between MAI and RBDQ-KR in the current study suggests that further studies are needed to support its plausibility. Instead, we have added the point that some of CCC values for alpha and theta bands tend to be lower in iRBD+P compared with control and/or iRBD groups, suggesting that this index might be useful to evaluate the disease progression. However, it was a cross-sectional study design; it would not possible to conclude whether the findings indicated a disease progression or not.Overall, future studies with longitudinal settings using these indices in larger sample sizes would be helpful to understand possible links between these diseases and to predict future development of degenerative diseases in iRBD patients.Further studies in longitudinal settings using these indices would be helpful to understand possible links between these diseases and to predict future development of degenerative diseases in iRBD patients.In conclusion, our findings indicate that CMC, CCC, and MAI based on PSG-EEG studies revealed distinct features in RBD with or without Parkinsonism. Furthermore, these indices may indicate a longitudinal biomarker useful to predict progression of Parkinsonism in iRBD patients with further validation based on the current observation of significant difference in iRBD and RBD-P groups.


For a long time, it was generally accepted that calcium influx is a necessary condition for ischemic injury mediated by NMDA receptors. However, recent studies have shown that NMDA receptor signaling, independent of ion flow, plays an important role in the regulation of ischemic brain injury. The purpose of this review is to better understand the roles of metabotropic NMDA receptor signaling in cerebral ischemia and to discuss the research and development directions of NMDA receptor antagonists against cerebral ischemia. This mini review provides a discussion on how metabotropic transduction is mediated by the NMDA receptor, related signaling molecules, and roles of metabotropic NMDA receptor signaling in cerebral ischemia. In view of the important roles of metabotropic signaling in cerebral ischemia, NMDA receptor antagonists, such as GluN2B-selective antagonists, which can effectively block both pro-death metabotropic and pro-death ionotropic signaling, may have better application prospects.Excessive activation of N-methyl- The divluN3A-B) .+, K+, or Ca2+ to enter or exit the cell and subsequently transmit ion-dependent excitatory signaling  receptors, and kainate (KA) receptors) is that glutamate binding opens ion channels, which allow Naignaling . Howeverignaling . The metignaling . With thignaling . Metabotignaling , synaptiignaling , dendritignaling  and longignaling . Recent ignaling . Metabotignaling  and presignaling . This mid-serine) jointly activate the receptor, initiating excitatory signaling. Unlike this classical mode, transduction of metabotropic NMDA receptor signaling only requires ligand binding to either one of the two agonist-binding sites, the one for glutamate, GluN2, or the one for glycine, GluN1 , but not 7CK  and calcium/calmodulin-dependent protein kinase II (CaMKII) bind to the intracellular C-terminus of NMDA receptors . Without2+, calmodulin, and protein kinase A (PKA) , and G-protein  . KA rece-protein . WhetherMetabotropic NMDA receptor actions involve signaling molecules, such as kinases, second messengers, and other molecules that have been found to be related to synaptic plasticity and cerebral ischemia .2+ influx following LTP induction, this signaling pathway promotes spine growth /nitric oxide synthase one adaptor protein (NOS1AP)/p38/MAPK-activated protein kinase 2 (MK2)/cofilin is a key metabotropic NMDA receptor signaling pathway for gating the structural plasticity of dendritic spines. nNOS is a member of the NMDA receptor complex that anchors to the scaffold protein postsynaptic density-95 (PSD-95) . NOS1AP e growth . It is se growth .PP1 and CaMKII are two important downstream signaling molecules of metabotropic NMDA receptor signaling involved in the process of synaptic depression. PP1 becomes an indirect coupling molecule of the GluN1 subunit by binding to yotiao . CaMKII p38 is also involved in synaptic depression mediated by metabotropic NMDA receptor signaling. NMDA exposure increased p38 phosphorylation in cultured neurons, which could be blocked by D-APV but not by MK-801 . Synapti2+-free extracellular solution with EGTA, MK-801, and strychnine  participates in the transduction of metabotropic NMDA receptor signaling. Co-incubation of hippocampal slices with metabotropic glutamate receptor type 5 (mGluR5) agonist CHPG (15 μΜ) and NMDA (5 μΜ) induced a robust increase in the phosphorylation level of ERK1/2, which could be inhibited by AP5, but not by MK-801 . This phrychnine . This efrychnine .The NMDA receptor, Src, and pannexin 1 (Panx1) comprise a metabotropic signaling complex that is involved in the process of cerebral ischemia . Src indAkt is another downstream metabotropic signaling molecule involved in cerebral ischemia. In a modified calcium-free extracellular solution with EGTA or BAPTA, treating mouse cortical neurons with glycine significantly enhanced the activity of Akt, which could be blocked by L-689560, but not by MK-801 or the glycine receptor antagonist, strychnine . After iIn addition to participating in the regulation of synaptic plasticity, p38 is involved in neuronal damage induced by cerebral ischemia. p38 activation induced by glutamate exposure or NO donors contributes to excitotoxic neuronal cell death . The nNO2+ influx, superoxide production was restored  activation requires both ionotropic and metabotropic NMDA receptor signaling. In mouse cortical neuron cultures, NMDA-induced superoxide production was blocked by the application of 7CK, L-689560, or MK-801, and after additional addition of ionomycin to provide a Carestored . Howeverrestored . NOX2 dorestored . After N of NOX2 .Metabotropic NMDA receptor signaling regulates the damage induced by cerebral ischemia in a bidirectional manner . In genein vitro  animals pretreated with MK-801 and strychnine; this effect was sensitive to glycine site antagonists and can also be blocked by Akt inhibitors . After iThe roles of NMDA receptors in cerebral ischemia are complex. NMDA receptors mediate both pro-death and pro-survival ionotropic signaling. Similarly, the metabotropic signaling of NMDA receptors can either be beneficial or harmful to neuronal survival. This makes the design of effective treatment strategies based on NMDARs difficult. The complexity of NMDA receptor signaling may be one of the important underlying reasons for the failure of NMDA receptor antagonists in the treatment of cerebral ischemia. Researchers should study how to effectively block all pro-death ionotropic and pro-death metabotropic signaling. Among all NMDA receptor antagonists, ion-channel blockers and glycine-binding site antagonists cannot block pro-death metabotropic signaling. Although glutamate-binding site antagonists can inhibit both ionotropic and metabotropic signaling, they have no selectivity for GluN2A and GluN2B. In theory, GluN2B-selective antagonists may have unique advantages for blocking the pro-death effect of both ionotropic and metabotropic signaling without influencing the pro-survival effect of GluN2A. However, existing GluN2B-selective antagonists are negative allosteric regulators and have the disadvantages of off-target effects and activity dependence . GluN2B-


The appropriate function of the BMP pathway depends on its interaction with other signaling pathways involved in neural differentiation, leading to synergistic or antagonistic effects and ultimately favorable biological outcomes. These opposite or cooperative effects are observed when BMP interacts with fibroblast growth factor (FGF), cytokines, Notch, Sonic Hedgehog (Shh), and Wnt pathways to regulate the impact of BMP-induced signaling in neural differentiation. Herein, we review the cross-talk between BMP signaling and the prominent signaling pathways involved in neural differentiation, emphasizing the underlying basic molecular mechanisms regarding the process of neural differentiation. Knowing these cross-talks can help us to develop new approaches in regenerative medicine and stem cell based therapy. Recently, cell therapy has received significant attention as a promising treatment for traumatic or neurodegenerative diseases. Therefore, it is important to know the signaling pathways involved in stem cell differentiation toward neural cells. Our better insight into the cross-talk of signaling pathways during neural development would improve neural differentiation within  The development of neurons is a multi-stage process that initiates early in embryogenesis that depends on several neural inducing factors  modulate transcription of BMP downstream target genes by translocation into the nucleus. In addition to the Smad-dependent canonical pathway, the BMP signaling pathway plays an essential role in cell proliferation and differentiation in a Smad-independent manner  through TAK1, MKK3, and p38 mitogen activated protein kinase  and Type II ; both types are serine and threonine protein kinases /protein kinase B (AKT), and PLPCγ pathways , which is essential for expressing Smad-interacting protein1 (Sip1) in the neural plate. As shown in Interleukin-6 (IL-6) family members such as leukemia inhibitory factor (LIF), cardiotrophin-1 (CT-1), and ciliary neurotrophic factor (CNTF) are among the central neuropoietic cytokines which regulate the proliferation, development, and differentiation of neural stem cells in different stages of neural development by recruiting the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway Bauer, . The JAKAstrocyte differentiation greatly depends on STAT3 activation and its binding to the promoter region of the target gene  formation of STAT3/Smad1/p300 transcriptional complex; and (2) a para-regulatory pathway between LIF and BMP2. In the first mechanism, LIF activates STAT3, while BMP2 triggers Smad1 through their specific receptors. As shown in It seems that all of the IL-6 family cytokines, which activate STAT3, potentially cooperate with BMPs to enhance astrocyte differentiation. The complex creation of Smad1 and STAT3 linked by p300 and para-regulatory pathways are suggested astrocyte differentiation mechanisms.via binding to the co-repressor complex, while the interaction of CSL and NICD induces basic helix–loop–helix (bHLH) transcription factors such as hairy and enhancer-of-split (Hes), Hey, Herp target genes  acts as a morphogenic factor in the patterning and cell-fate specification of the central nervous system. During the development of the mammalian CNS, Shh regulates the proliferation, differentiation, and survival of neural stem/progenitor cells Komada, . Shh indMany signaling factors have been implicated in regulating dorsoventral (DV) patterning during embryonic CNS development, among which BMP and Shh signalings play essential roles. Shh factors are derived from the floor plate and notochord, while BMPs are secreted from the ectoderm and roof plate  to exit from the cell cycle and enter the differentiation program. This inhibitory effect may be conducted through the suppressive impact of BMP on transcriptional factors of Shh such as Smo and Gli1, allowing granule neuron differentiation  differentiation toward oligodendroglial and astrocytic, respectively , or non-canonical , casein kinase 1(CKIα), and GSK3 is functional. In the intact form of the destruction complex, GSK3 displays enzymatic activity that leads to phosphorylation of β-catenin and resultant degradation by the proteasome system. As shown in Many studies focused on the role of Wnt signaling in adult neurogenesis. Activation of the Wnt signaling pathway enhances hippocampal neurogenesis and the differentiation of NPCs into mature neurons in Alzheimer’s disease (Oh et al., There is cross-talk between Wnt and BMPs signalings in nervous system development through functional implications in many processes. BMP and Wnt signalings synergistically or antagonistically interact at three levels: First, BMP and Wnt regulate ligand production crucial for creating intracellular response of these morphogens during embryonic development. Second, the occurrence of cytoplasmic interactions between components of BMP/Wnt pathways. Third, they share some target genes in the nucleus (Ille et al., Wnt and BMP can intricately regulate each other through synergistic signaling and feedback systems during nervous system development (Itasaki and Hoppler, On the other hand, Wnt and BMP signaling can antagonize different processes, such as neural and neuroepithelial cell development (Feigenson et al., In addition to the agonistic and antagonistic interaction of BMP and Wnt signaling, they can act as upstream or downstream effectors for each other. Wnt signaling can act upstream of BMP and upregulate its pathway in some developmental systems, such as neurogenesis and astrogliogenesis. Conversely, BMP may upregulate and act upstream of Wnt signaling in biological functions such as neural crest delamination and dorsal/ventral patterning (Fuentealba et al., Several signaling pathways regulate nervous system development during different stages of fetus formation. These signaling pathways cooperate in a synergistic or antagonistic manner that forms various signaling cross-talks. BMPs pathway as a pivotal signaling in neural development interacts with other signaling, including FGF, cytokines, Notch, Shh, and Wnt/β-catenin. FGF can increase neural differentiation by inhibiting Smads, a downstream protein in BMP signaling, mainly by inserting phosphorylations in inhibitory regions of Smads. On the other hand, cytokines increase astrogenesis in a synergistic manner with BMPs that include enhancing transcription complex and/or a para-regulatory pathway. Similarly, Notch cooperates with BMPs to increase gliogenesis based on the expression pattern of bHLH family members. Besides, Shh and Wnt increase neural differentiation by affecting Smads and/or downstream proteins of BMP signaling. In view of the complex interaction among different cellular pathways, considering the cooperation mechanisms of the BMP pathway with the other signalings can improve the pre-clinical and translational experience of using stem cells as a regenerative agent in treating nervous system damages; an issue which requires further investigation in the future studies.AM-T, AB, and HN: conceptualization. AM-T and AB: investigation and writing—original draft preparation. AM-T, AB, MF, and MH: writing—review and editing. MF: drawing figures. HN and SB: supervision. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


A quantitative study was carried out to compare the proteome of peripheral blood mononuclear cells (PBMCs) of vaccinated goat and sheep [5 days post-vaccination (dpv) and 14 dpv] vs. unvaccinated (0 day) to divulge the alteration in protein expression following vaccination. A total of 232 and 915 proteins were differentially expressed at 5 and 14 dpv, respectively, in goats. Similarly, 167 and 207 proteins were differentially expressed at 5 and 14 dpv, respectively, in sheep. Network generated by Ingenuity Pathway Analysis was “infectious diseases, antimicrobial response, and inflammatory response,” which includes the highest number of focus molecules. The bio functions, cell-mediated immune response, and humoral immune response were highly enriched in goats at 5 dpv and at 14 dpv. At the molecular level, the immune response produced by the PPRV vaccine virus in goats is effectively coordinated and stronger than that in sheep, though the vaccine provides protection from virulent virus challenge in both. The altered expression of certain PBMC proteins especially ISG15 and IRF7 induces marked changes in cellular signaling pathways to coordinate host immune responses.In the present study, healthy goats and sheep ( Peste des petits ruminants (PPR) is a severe, contagious viral disease of small ruminants, mainly sheep and goats, caused by peste des petits ruminants virus (PPRV) belonging to the genus Morbillivirus and family Paramyxoviridae  play a vital role in the immune response  and havefections , 13, 14.in PBMCs  and viruin PBMCs , 16. To Vaccination of sheep and goats with live attenuated virus is shown to elicit protective immunity to infection with virulent PPRV; yet, the mechanisms that induce immune response and confer protection from virulent PPRV strains are still not completely clear. In this study, we examine the proteome changes that occur in PBMCs of sheep and goat in response to PPRV vaccination, which contribute to the development of immunity, by identifying differentially expressed proteins (DEPs) and biological pathways associated at the early time points. To establish differences in vaccine response in sheep and goat, DEPs in PBMCs at 5 and 14 dpv in comparison to 0 dpv as control were identified. Thereafter, we performed extensive pathway and network analyses to find out differences in the underlying protein pathways associated with PBMCs of sheep and goat at different time points.n = 5) and sheep (n = 5) confirmed negative for PPRV antibodies by monoclonal antibody-based competitive ELISA  were vaccinated, and PBMCs were isolated from the animals before (0 day) and 5 and 14 days post-vaccination . Thereafter, proteome profiling of PBMCs isolated at the different time points, 0 day, 5 dpv, and 14 dpv, was carried out. DEPs at 5 and 14 dpv were identified, respectively, by comparing the proteome data of 5- and 14-dpv PBMCs with 0-day PBMC data.Goats and sheep -based competitive-ELISA test . The sam7 cells of PBMCs were taken in lysis buffer [50 mM Tris Buffer + protease inhibitor cocktail (PIC) + phenylmethylsulfonyl fluoride (PMSF)], homogenized on ice, centrifuged at 14,000 rpm, and the supernatant was collected into a separate tube (Tris Buffer extract). Urea lysis buffer was added to the cell pellet. This was followed by centrifugation at 14,000 rpm and collection of the resultant supernatant. For quality check, the supernatant was run on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentration was determined using the Bradford assay, and 100 μg of the samples was taken for digestion.The PBMC subset isolation was carried out following the standard procedure as described in our previous study . For proThe samples were added with 100 mM dithiothreitol (DTT) for 1 h at 95°C, treated with 250 mM iminodiacetic acid (IDA), and kept for 45 min at room temperature in the dark. This was followed by digestion of the sample with trypsin and overnight incubation at 37°C. Thereafter, formic acid (1%) was added to it, incubated at 37°C for 1 h and vacuum dried. The resulting sample was dissolved in 10 μl of 0.1% formic acid and centrifuged at 10,000 × g. The supernatant was injected on C18 Nano-LC column for separation of peptides. The Waters Synapt G2 Q-TOF instrument for MS and MS/MS were used for analysis.Through MassLynx 4.1 Waters, the raw data were processed, and through PLGS software, the proteins were identified. The threshold while considering a protein was as follows: Minimum number of peptides to be found for a protein, 2; Minimum number of fragment (MSMS) ions in a peptide, 3; Minimum number of fragment (MSMS) ions in a protein, 7; Peptide mass tolerance, 30 ppm; and Fragment ion mass tolerance, 70 ppm. The quantification of the protein was carried out through Expression analysis package of the PLGS software, and the identified proteins in the three runs of each sample were compared with each other as control  and vaccinated samples.p-values (Fisher's exact test) and activation z-scores were calculated by IPA. Core analysis and comparison analysis for each dataset were performed to know significant (p < 0.05) and activated (Z score > 2) or inactivated (Z score < −2) canonical pathways. Network analysis was also performed.Data were analyzed using QIAGEN's IPA . The uniport ID of DEPs was converted into gene ID from uniport data set, and this list of genes with corresponding fold change was uploaded to IPA for downstream analysis. The canonical pathways and functional processes of biological importance that were the most significant were identified using the list of DEPs identified with liquid chromatography–mass spectrometry (LC-MS) and the Ingenuity Pathways Knowledge Base (IKB). Pathway enrichment In this study, sheep and goats screened -ve for PPRV antibodies by competitive-ELISA and SNT and for PPRV antigen by s-ELISA were used. Vaccinated goats were found seropositive by 9 dpv, while sheep had an idiosyncratic response in the range of 9–14 dpv for different animals .A quantitative study was carried out to compare the proteome of PBMCs of vaccinated goat and sheep (5 and 14 dpv) vs. unvaccinated (0 day). A total of 232 and 915 proteins were differentially expressed at 5 and 14 dpv, respectively, in goats. Similarly, 167 and 207 proteins were differentially expressed at 5 and 14 dpv, respectively, in sheep  at a cutImmune system processes enriched in goats at 5 dpv were type I interferon signaling, cytoplasmic PRR signaling pathway in response to virus, interferon gamma-mediated signaling pathway, regulation of innate immune response, and regulation of adaptive immune response , whereasp < 0.05) and activated (Z score > 2) or inactivated (Z score < −2) canonical pathways. In vaccinated goats at 5 dpv, all the pathways were found to be activated (Z score > 2) like dendritic cell maturation, TREM1 signaling, nuclear factor (NF)-κB signaling, phosphoinositide 3-kinase (PI3K) signaling in B lymphocytes, etc. Dendritic cell maturation pathway was the most significantly enriched  pathway. The ratio—number of the proteins in the dataset mapping to each specific pathway divided by the total number of proteins in the pathway—was highest for TREM1 pathway  pathway with highest ratio also  pathway, and IL-9 signaling pathway had the highest ratio  or inactivated (Z score < −2) . StimulaIn vaccinated goats at 5 dpv, 19 networks were generated, out of which networks 3, 9, 11, and 15 were overlapping . The netp-value) enriched in goats than in sheep. Dendritic cell maturation, TREM1, NF-κB signaling, icos-icosL signaling in T helper cells, PI3K signaling in B lymphocytes, CD27 signaling in lymphocytes, etc., showed higher enrichment in goats, whereas in sheep, the role of nuclear factor of activated T cells (NFAT) in the regulation of the immune response was highly enriched  signaling pathways, innate immune response, inflammatory response, positive regulation of signal transduction and cytokine production, RIG signaling pathway, chemokine signaling pathway, Class-I major histocompatibility complex (MHC) presentation and processing, and classical antibody-mediated complement activation (A quantitative study carried out to compare the proteome of PBMCs of vaccinated goat and sheep (5 and 14 dpv) vs. control (0 dpv) revealed enrichment of immune system processes and orchestration of antiviral state by type I interferon signaling, cytoplasmic PRR signaling pathway in response to virus, interferon gamma-mediated signaling pathway, regulation of innate immune response, regulation of adaptive immune response, negative regulation of T-cell activation, regulation of type 2 immune response, positive regulation of B-cell proliferation. From transcriptome analysis of PBMCs in tivation , 10, 25.ISG15, Mx1, Mx2, RSAD2, IFIT3, and IFIT5) and transcription factors like IRF-7 and STAT-1 that induce ISG expression were found in lymphocytes during virulent PPRV infection , R1G-1 (DDX58), and ISGS  enriched in goats than in sheep. Dendritic cell maturation, TREM1 signaling, NF-κB signaling, ICOS-ICOSL signaling in T helper cells, PI3K signaling in B lymphocytes, CD27 signaling in lymphocytes, etc., showed higher enrichment in goats. TREM1 regulates T-cell proliferation and activates antigen-presenting cells  under the Committee for Control and Supervision of Experiments on Animals(CPCSEA), India.RS, BPM, and RG conceived and designed the research and proofread the manuscript. KR and DM performed the vaccine testing experiment. SW, ARS, and SS conducted the wet lab work. SW, ARS, RK, MP, and RG analyzed the data. SW, ARS, RK, AS, and BM helped in manuscript drafting and editing. All authors contributed to the article and approved the submitted version.This study was supported in part by the Centre for Agricultural Bioinformatics (ICAR-IASRI) (CABin/100644/16103/801/10133) and SubDIC (BTISnet), ICAR-IVRI.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Almost three decades after its seminal discovery, our understanding of the remarkable TOR pathway continues to expand. As a TOR complex, TORC2 lies at the nexus of many signaling pathways and directs a diverse array of fundamental processes such as cell survival, proliferation, and metabolism by integrating environmental and intracellular cues. The dysregulation of TORC2 activity disrupts cellular homeostasis and leads to many pathophysiological conditions. With continued efforts at mapping the signaling landscape, the pace of discovery in TORC2 regulation has been accelerated in recent years. Consequently, emerging evidence has expanded the repertoire of upstream regulators and has revealed unexpected diversity in the modes of TORC2 regulation. Multiple environmental cues and plasma membrane proteins that fine-tune TORC2 activity are unfolding. Furthermore, TORC2 signaling is intricately intertwined with other major signaling pathways. Therefore, feedback and crosstalk regulation also extensively modulate TORC2. In this context, we provide a comprehensive overview of revolutionary concepts regarding emerging regulators of TORC2 and discuss evidence of feedback and crosstalk regulation that shed new light on TORC2 biology. In eukaryotes, the evolutionarily conserved Ser/Thr kinase target of rapamycin (TOR) functions as a central integrator when growth factors, nutrients, and cellular energy status favor anabolism. These stimuli activate TOR metabolic pathways and ultimately drive cell growth and survival . In mammTORC2 exerts pleiotropic effects on cellular metabolism and homeostasis primarily through activation of downstream effectors. Therefore, TORC2 activity is defined as its ability to phosphorylate downstream targets, which are currently well recognized as AGC kinases, including Akt, PKC, and SGK1 . The mosFor proper maintenance of cellular homeostasis, the activation of TORC2 should be precisely regulated inside the cell. There are many indicators that dysregulation of TORC2 activity underlies a wide spectrum of human pathologies, including cancer, metabolic disorders, autoimmune diseases, aging, and neuronal-related diseases . TherefoPartially due to the lack of acute pharmacological agents that tease apart the two TOR complexes, the regulation of TORC2 is relatively less studied and still poorly defined compared to that of TORC1. However, emerging evidence in recent years has greatly expanded the repertoire of upstream regulators of TORC2. It is becoming clear that, unlike TORC1 activity that mainly depends on lysosomes , the regIn this review, we focus on current major developments in TORC2 regulation that illustrate how the expanding spectrum of emerging inputs influences its activity. We also discuss how TORC2 signaling is rewired and intersects with other major pathways through feedback and crosstalk regulation, which also profoundly modulate TORC2 signaling.TORC2 activity is tightly associated with the plasma membrane. Although the precise subcellular localization of TORC2 is still under debate, at least some TORC2 in both mammalian cells and yeast appears to be on the plasma membrane as detected by microscopy and biochemical fractionation approaches . Therefoin vitro mTORC2 activity, using Akt-S473 as a substrate, are stimulated by growth factors  gets phosphorylated . In thisRecent biochemical labeling and imaging studies using a compartment-specific mTORC2 activity reporter, dubbed LocaTOR2, revealed an alternative mechanism for mTORC2 activation. In mammalian cells, the activity and localization of mTORC2 via mSin1-PH at the plasma membrane is PI3K- and growth factor-independent, and membrane recruitment of Akt is sufficient for mTORC2-mediated Akt-S473 phosphorylation in response to growth factors . This isThe majority of early studies suggest that mTOR is not regulated by Ras through direct contact but, rather, distally via Ras stimulation of PI3K and mitogen-activated protein kinase (MAPK) pathways . HoweverOur recent study identified an essential cell adhesion receptor CD146 that directly linked mTORC2 activation with extracellular growth factors to enable cell proliferation and survival . The actThe observation that mTORC2 activation depends on its association with juxtamembrane positively charged amino acid cluster of CD146 might represent a general mechanism for the regulation of mTORC2 activity at the plasma membrane. It should be noted that several Ras proteins also contains a similar polybasic motif in their juxtamembrane hypervariable region , which mIn endothelial cells, mTORC2 was observed localized to plasma membrane raft domains. Syndecan-4, which is a single-pass transmembrane proteoglycan, recruits PKCα to the membrane raft to regulate PKCα activity; this, in turn, is required for appropriate mTORC2 localization to rafts and subsequent phosphorylation of Akt, PKC, GSK-3, eNOS, and Foxo1/3a in response to FGF2 . This prMembrane tension is generated as a response to any application to the membrane surface of external forces . LocalizThe regulation of TORC2 activity by membrane tension is conserved in mammalian cells. Vertebrate-specific membrane invaginations called caveolae has been suggested to function analogously to eisosomes. Mechanical stretch, which leads to rapid disassembly of caveolae, triggers mTORC2-dependent phosphorylation of Akt-S473 . This prThe engagement of Toll-like receptors (TLRs) by their cognitive ligands [such as lipopolysaccharide (LPS)] acutely activates mTORC2 as indicated by the phosphorylation of Akt, PKCα/β, SGK1, NDRG1, and Foxo1/3, and mTORC2 negatively feedbacks to TLR signaling to suppress the pro-inflammatory response in multiple types of mammalian cells . HoweverTORC1 activity is mainly dependent upon sensing amino acids and tightly regulated by other nutrient inputs to support cell metabolism and growth throughout evolution; TORC2 activity is not as tightly coupled to nutrient availability. In recent years, emerging evidence has suggested that TORC2 activity also responds to and can be modulated by distinct nutrients and metabolites. However, the robustness and pathophysiological implications will require further validation.TORC2 activity is regulated by glucose. In human glioblastoma, for example, extracellular high glucose conditions activate mTORC2 (as indicated by Akt-S473 and NDRG1-T346) via acetyl-CoA-dependent acetylation of Rictor. Rictor acetylation is maintained to form an autoactivation loop of mTORC2 even in the absence of growth factor receptor signaling , nominatAmino acids can both activate and inhibit TORC2 activity depending on the cellular context. For example, amino acids positively regulate mTORC2 activity (as indicated by increased phosphorylation of Akt-S473 and GSK-3), together with mTORC1, via class I PI3K and only mTORC2 is required for purine synthesis in multiple human cells . On the TORC2 activity (together with TORC1) responds to folate stimulation without a clear mechanism . Folate Saccharomyces cerevisiae, methylglyoxal activates TORC2-PKC as well as TORC2-Akt signaling  is an energetic stress kinase that promotes catabolic and suppresses anabolic metabolism coordinately to restore energy balance . Given sGTPases play essential roles in regulating TORC2 activity. Rictor and mSin1 contain RasGEFN  and Ras binding domain, suggesting a correlation with GTPase . Indeed,ROS functions as an TORC2 activating factor in protecting cells from oxidative stress from yeast to mammalian cells. In yeast, ROS can activate TORC2 through an unknown mechanism and in turn, TORC2 regulates ROS metabolism to control cell growth and survival . The actA hallmark of signaling networks is the presence of multiple nodes with feedback loops. The TORC2 signaling network provides many typical examples of feedback control, a common feature of cellular signaling systems. Similar to other signaling pathways, TORC2 signaling is subjected to negative feedback regulation, which ensures switch-like behavior from initiation of stimulatory signals to termination of the signaling relay in a spatially and timely controlled manner. Therefore, several downstream signals of TORC2 function as negative regulators as TORC2 signaling. Besides negative feedback regulation, TORC2 is also regulated by positive feedback, which acts as a booster for TORC2 signaling .Feedback of TORC2 signaling is regulated at the level of TORC2-IRS (insulin receptor substrate). TORC2 responds to insulin signaling through IRS-mediated PI3K activation . TORC2 cAmong mTORC2 downstream effectors, mTORC1 functions as a negative feedback regulator that acutely inhibits mTORC2 signaling through several distinct mechanisms in mammalian cells. First, short-term treatment with rapamycin enhances the activity of Akt in response to insulin and IGF1, suggesting mTORC1 inhibits mTORC2 signaling via feedback regulation between mTORC1 and insulin/PI3K signaling . Indeed,Akt exerts both positive and negative feedback regulation of mTORC2 signaling depending on distinct phosphorylation of mTORC2 components. In human cells, the mTOR protein is phosphorylated at T2173 of the ATP-binding site in the kinase domain in an Akt-dependent manner, which impairs mTORC2 activity; this functions as a negative feedback to directly control mTORC2 signaling . In contThe role of FOXO in the feedback regulation of TORC2 signaling comes from cancer studies. In some cancer cells, prolonged inhibition of Akt signaling relieves feedback suppression of RTK expression and activity through FOXO-dependent transcriptional induction, thereby enhancing PI3K-Akt signaling . On the In addition to the independent signaling program of TORC2 that provides compensatory mechanisms, the TORC2 signaling network is extensively intertwined and has several points of crosstalk with other major signaling pathways. These signaling pathways, which include Hippo, WNT, and Notch, act both upstream and downstream of TORC2 pathways .There is a particularly intimate relationship between the TORC2 and Hippo tumor-suppressor pathways, which engage in bilateral cross-regulation at multiple levels. The Hippo pathway determines precise control of cell size by inhibiting phosphorylation of the transcription co-activator Yes-associated protein (YAP). Upon Hippo inactivation, the active and hypophosphorylated YAP translocates into the nucleus to trigger the transcription of genes encoding cell proliferation . It has The bilateral cross-regulation of Hippo and mTORC2 is also mediated by Mst1 kinase, another key component of the Hippo pathway. Mst1 has been shown to phosphorylate and inactivate FOXO1, substrates of mTORC2-Akt, and thereby regulates mTORC2 downstream signaling and cell death in neuronal cells . In cardAnother major signaling pathway that exhibits cross-regulation with TORC2 is the WNT pathway. During osteoblast differentiation, the non-canonical ligand WNT3A activates mTORC2 and therefore induces aerobic glycolysis known as the Warburg effect. This mTORC2-mediated glucose metabolism requires the binding of WNT3A with its co-receptor low-density lipoprotein receptor-related protein 5 (LRP5) but not canonical WNT signaling. Moreover, WNT3A-mediated mTORC2 activation requires RAC1 . The mTOThe Notch pathway is another example of crosstalk with TORC2 signaling. Notch plays critical roles in the control of proliferation and cell fate decisions . Notch rExciting progress has been made over the last few years in elucidating the complex network of TORC2 signaling and its broad implications in human physiology and pathology. Basic investigations into the genetics, cellular biology, biochemistry, and structural biology of TORC2 have yielded a sophisticated view of how the activity of this multi-protein kinase is regulated. As discussed above, TORC2 acts as a central signaling node that is precisely and exquisitely regulated by a variety of upstream inputs to exert widespread control over many biological processes and maintenance of cellular homeostasis. A natural question is why TORC2 has evolved such diversified modes of regulations. To this end, it is helpful to first consider the biological functions of TORC2, as it centrally controls many fundamental requirements and processes for life, such as cell proliferation, survival, and metabolism. In principle, any perturbation or interference with these processes might converge on this kinase and profoundly impact its activity. Another possible explanation might lie in the molecular composition and structural features of TORC2. TORC2 is a multi-protein kinase consisting of multiple accessory subunits, such as Rictor and Sin1, in addition to the catalytic subunit TOR. These components are large and possess a number of regulatory domains, interactive interfaces, and modifiable sites. These unique compositional and structural features allow TORC2 to respond to a wide array of inputs, and at the same time, permit interactions and post translational modifications that fine-tune TORC2 integrity, assembly, or, perhaps substrate recruitment. Thirdly, like many other protein kinases, the activity of TORC2 heavily relies on its subcellular localization, especially to membranous structures. Therefore, signals that guide TORC2 distribution to specific cellular compartments could also extensively modulate its activity, which permits the sensing of TORC2 to diverse inputs or allow access to distinct substrates at the right time and at the right place. Last but not least, TORC2 signaling has multiple connecting nodes that allow both compensatory feedback control from within TOR signaling, as well as crosstalk with other signaling pathways. These new connections have significantly complicated the regulation of TORC2 activity and its possible biological impacts.The identification of many emerging upstream inputs and feedback as well as crosstalk mechanisms is revealing the broad scope of TORC2 regulatory modes and is also raising interesting and challenging questions about the regulation of this complex. Signals generated at the plasma membrane are emerging regulators for TORC2 signaling. However, the regulatory mechanisms governing TORC2 activation at the plasma membrane are still incompletely understood. For example, are growth factors and PI3K signaling indeed required for TORC2 activation, and to which extent? How exactly do they control TORC2 membrane targeting and activation? How does membrane tension act on the plasma membrane to regulate TORC2 activation in mammalian cells? Does it involve the dynamics of the cytoskeleton? Could other exogenous agents, such as viral or bacterial pathogens signal TORC2 through their cognitive receptors in a host cell? How do metabolites and extracellular nutrients regulate TORC2 activation at the plasma membrane? Could this pathway be explored for the development of new therapies for metabolic disorders and cancer?Compared with the well characterized lysosome localization of TORC1 activity, the localization of TORC2 is more ambiguous. There could be multiple pools of TORC2, each at a distinct subcellular location. Indeed, TORC2 localization and activity have been observed at several specialized membranous structures, including mitochondria, endosomes, endoplasmic reticulum (ER), and mitochondria-associated ER membrane (MAM), in addition to the plasma membrane . TherefoDysregulation of TORC2 activity has been linked to many disease processes; this has been firmly established in metabolic diseases and cancer. Efforts are underway to develop selective strategies for targeting TORC2 that are directly involved in disease progression. Because specific and acute small-molecule inhibitors of this kinase are currently unavailable, the identification of TORC2-targeted strategies should be facilitated by the availability of novel mechanisms of TORC2 regulation. One possible approach for TORC2 selective inhibition will be via disruption of complex-specific protein-protein interactions, e.g., the association of mTORC2 with CD146 or other membrane proteins. These targeting approaches would not affect other signaling of TOR pathways or TORC1-dependent negative feedback loops. Alternatively, the rational design of TORC2-specific inhibitors might also be facilitated by the availability of a high resolution 3D structure of TORC2 . The ideAs these ongoing mysteries being revealed and our understanding of these questions becomes increasingly sophisticated, it will not only advance our current view of many vital physiological processes but also provide an important context for therapeutically countering the effects of pathogenic TORC2 in cancer and many other diseases, ultimately improving the quality of human life.JL and YL conceived the concept of article. PA, WX, JL, and YL wrote the manuscript. WX and YL prepared all the figures. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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