The mechanistic target of rapamycin (mTOR) is a master regulator of cell growth, proliferation, and metabolism by integrating various environmental inputs including growth factors, nutrients, and energy, among others. mTOR signaling has been demonstrated to control almost all fundamental cellular processes, such as nucleotide, protein and lipid synthesis, autophagy, and apoptosis. Over the past fifteen years, mapping the network of the mTOR pathway has dramatically advanced our understanding of its upstream and downstream signaling. Dysregulation of the mTOR pathway is frequently associated with a variety of human diseases, such as cancers, metabolic diseases, and cardiovascular and neurodegenerative disorders. Besides genetic alterations, aberrancies in post-translational modifications (PTMs) of the mTOR components are the major causes of the aberrant mTOR signaling in a number of pathologies. In this review, we summarize current understanding of PTMs-mediated regulation of mTOR signaling, and also update the progress on targeting the mTOR pathway and PTM-related enzymes for treatment of human diseases. The mechanistic target of the rapamycin (mTOR) pathway is a central sensor of environmental clues, such as growth factors, nutrients, and energy, through which it controls proper cell growth and metabolism. Imbalances in the mTOR pathway including full inhibition or constitutive activation may disrupt the cellular homeostasis, leading to human diseases including but not limited to cancers, metabolic diseases, and aging and neurodegenerative disorders ,2,3. In mTOR is an evolutionarily conversed protein kinase from yeast to human. In mammals, mTOR is the catalytic subunit of two different complexes , termed mTORC1 exerts its biological functions by phosphorylating a large number of downstream proteins. mTORC1 phosphorylates S6K and 4E-BP1 to promote protein synthesis . mTORC1 mTORC1 activation is spatially and temporally controlled by upstream inputs, notably amino acids and growth factors. In response to amino acids stimulation, Rag GTPases (Rags) are recruited to the lysosome by the Ragulator complex  ,20. SubsUnlike mTORC1, mTORC2 is insensitive to rapamycin and has distinct roles on metabolism, cytoskeleton regulation, cell proliferation, and survival ,11,37. THaving discovered dozens of proteins in the mTOR pathway, it is important to understand how these components are regulated, which may provide explanations for the aberrant mTOR signaling in human diseases. Besides genetic alterations and transcriptional regulations, post-translational modifications (PTMs) including phosphorylation, ubiquitination, acetylation, and glycosylation have been demonstrated to be key regulators of mTOR signaling. In this review, we will summarize PTMs-mediated regulation of the mTOR pathway  and discStudies have demonstrated that mTOR kinase activity can be regulated by several types of PTM, notably phosphorylation. Growth factors promote the phosphorylation of mTOR at S2448 by Akt or S6K1 ,44,45,46mLST8 knockout mice are defective in vascular development [As the shared subunit of the mTORC1 and mTORC2 complexes, mLST8 was initially identified to bind to the mTOR kinase domain and stabilize mTOR interaction with Raptor, leading to enhanced mTORC1 activity toward S6K1 and 4E-BP1 . Howeverelopment , indicatβ-TRCP, resulting in its ubiquitination and degradation [DEPTOR, also known as DEPDC6, is an mTOR-interacting protein that serves as an endogenous inhibitor of both mTORC1 and mTORC2. DEPTOR depletion enhances mTOR signaling, cell growth, and survival. In contrast, DEPTOR overexpression blocks mTORC1 activation but unexpectedly activates mTORC2 signaling through relieving the mTORC1-mediated inhibitory feedback signal to the PI3K/mTORC2/Akt pathway . Generalradation ,65,66. Mradation . These sRaptor in mouse leads to various tissue-specific diseases, such as heart failure [Raptor-knockout promotes liver cancer development [Raptor is the unique subunit of the mTORC1 complex and acts as a scaffolding protein to determine mTORC1 activity and substrate specificity ,5,9,10.  failure , hepatic failure , and mus failure . Unexpecelopment . Similarelopment . JNK, RSelopment ,75,76,77elopment . Recentlelopment . Other Pelopment ,81. Sungelopment .In 2007, three independent groups identified PRAS40 as a Raptor-interacting protein that acts as an inhibitor to block insulin/Rheb-mediated activation of mTORC1 signaling ,84,85. ITSC complex, composed of TSC1, TSC2, and TBC1D7, is a negative regulator of mTORC1 signaling . MutatioRheb is a small GTPase that is enriched on multiple endomembrane compartments including the lysosomal membrane where it interacts with and activates mTORC1. Farnesylation of Rheb in the C-terminal CaaX motif is required for its lysosomal localization ,106. In GTP hydrolysis, leading to inactivation of mTORC1 signaling [RNF152 knockout leads to hyperactivation of mTORC1 [The activation of the Rag heterodimer is the key step for mTORC1 lysosomal recruitment and activation. Notably, RagC is frequently mutated in follicular lymphoma  and the ignaling ,115. Howf mTORC1 . In contf mTORC1 .Ragulator interacts with Rag GTPases and is necessary for localizing the Rag proteins and mTORC1 to the lysosomal surface for activation. Among the five components of the Ragulator complex, myristoylation and palmitoylation in the N-terminus of p18 serve as a membrane localization signal for tethering Ragulator to the lysosomal surface . MoreoveThe GATOR1 complex is composed of three subunits including DEPDC5, NPRL2, and NPRL3, while the GATOR2 complex consists of WDR24, WDR59, MIOS, SEH1L, and SEC13 . KISCTORFLCN leads to cardiac hypertrophy and dysfunction in mice [GTP hydrolysis in the presence of amino acids [FLCN is considered as a tumor suppressor, mutations in which are the drivers of Birt–Hogg–Dubé hereditary cancer syndrome . Moreove in mice . FLCN funo acids . FLCN wano acids , S406/S5no acids , and S30no acids . FLCN hano acids ,132. HowTo date, three types of cytosolic amino acid sensors for the mTORC1 pathway have been identified: the leucine sensor Sestrin2, arginine sensor CASTOR1, and S-adenosylmethionine (SAM) sensor SAMTOR . MechaniAs a S/T protein kinase, mTORC1 exerts its biological functions on cell growth and metabolism by phosphorylating multiple downstream substrates, most of which have been demonstrated to play a role in various types of human diseases, such as cancers and cardiovascular and neurodegenerative diseases . Here, wThe S6K kinases (S6K1 and S6K2) are critical downstream effectors of mTORC1 in controlling fundamental cellular processes, such as transcription, translation, metabolism, and cell growth . PhosphoULK1, a gate keeper for autophagy initiation, is a key downstream effector mediating the role of mTORC1 signaling in autophagy. Upon glucose starvation, AMPK activates ULK1 by directly phosphorylating S317/S377 to induce autophagy. Conversely, under nutrient- and energy-rich conditions, mTORC1 directly phosphorylates ULK1 at S757 to disrupt ULK1 interaction with AMPK, leading to autophagy inhibition . RecentlGrb10 is an adapter protein that interacts with tyrosine kinase receptors, such as insulin receptor . mTORC1-In searching the mTOR-interacting proteins, Rictor was identified as a novel mTOR-associated component, which also binds to mLST8 and confers a rapamycin-insensitive feature to mTORC2 . AcetylaLater studies characterized Sin1 as another essential component of the mTORC2 complex, the depletion of which abolishes mTORC2 complex formation ,158. SinIt has been reported that the Sin1-PH domain binds to PIP3 and subsequently releases its inhibition on the mTOR kinase domain, leading to mTORC2 activation in response to growth factors . Thus, tGDP at S192 by GSK3 mediates its interaction with RasCGTP to promote mTORC2 activation [Like mTORC1, several small GTPases have been identified recently to directly regulate mTORC2 kinase activity. The Rho family of GTPase Rac1 directly binds to mTOR through the C-terminal region and promotes the activity of both mTORC1 and mTORC2 independent of its nucleotide binding states and PI3K pathway . Of the tivation . These sAs a major substrate, Akt is phosphorylated at S473 by mTORC2 , which cBesides Akt, PKCs and SGK1 are also the downstream effectors of mTORC2. Phosphorylation of PKCα by mTORC2 in the turn motif promotes its maturation, stabilization, and activation, which controls actin polymerization, cell shape, and mobility ,183,184.Dysregulation of mTOR signaling is tightly associated with many human diseases, such as obesity, diabetes, cancers, and neuronal disorders , making Despite TORKi being able to block mTORC2-dependent phosphorylation of Akt-S473, its inhibition on mTORC1 may release the negative feedback regulation of PI3K/PDK1/Akt signaling ,198, conAs the major downstream effector of PI3K/mTOR signaling, Akt plays a central role in promoting cell survival and apoptosis. Given hyperactivation of Akt is a hallmark of cancers, targeting Akt for cancer therapy has been extensively investigated . Many AkAs the key events in signaling transduction, PTMs including phosphorylation, ubiquitination, methylation, and acetylation, among others, provide a tight and reversible regulation of mTOR signaling. Aberrancies in these PTM-related enzymes may lead to the aberrant activation of the mTOR pathway. Therefore, targeting these mTOR-related regulators would offer an option for treating mTOR-associated human diseases. Below, we primarily focus on kinases, E3 ubiquitin ligases, and deubiquitinases, which are key regulators of the mTOR pathway.As S/T kinases, RSKs regulate diverse cellular processes by phosphorylating many downstream substrates . NotablyJNKs including JNK1, JNK2, and JNK3 in mammals are predominantly activated by environmental stress and regulate many physiological processes, such as cell proliferation, apoptosis, immune response, inflammation, and neuronal plasticity . Their hThe PIM kinase family containing three members, PIM1–3, and plays a critical role in cell proliferation, apoptosis, and migration by directly phosphorylating a number of downstream targets, such as MYC, CDC25, p21/p27, and TSC2. Overexpression of PIM kinases has been observed in a variety of human cancers and is associated with poor prognosis and drug resistance in prostate cancer and breast cancer . As therOther mTOR pathway-associated kinases including cdc2 , IKKβ 2, AMPK 2, LATS 2, ULK1 2, and GSKAs described above, a dozen E3 ligases and DUBs have been identified to regulate mTOR signaling by controlling protein stability, enzymatic activity, and complex formation. Aberrancies in these E3s/DUBs are frequently observed in human diseases, which may be in part responsible for the dysregulation of mTOR signaling. FBXW7 mutations [FBXW7, a cullin-based SCF type of E3 ubiquitin ligase, plays an important role in various biological processes such as cell cycle transition, cell proliferation, apoptosis, metastasis, and immune response . FBXW7 iutations . Beside Parkin belongs to the RING-between-RING (RBR) family of E3 ubiquitin ligases that regulate a variety of cellular processes including mitochondrial quality control, anti-oxidative stress, and mitophagy. Mutations in Parkin lost its protective role on the survival of dopaminergic neurons, which is the major cause of autosomal recessive parkinsonism. Notably, mTOR signaling exerts either a neuroprotective or neurotoxic role depending on PD models and has been linked to PD. It will be interesting to investigate whether Parkin-mediated ubiquitination and activation of mTOR signaling play a role in PD. Moreover, Parkin is an emerging tumor suppressor as it is frequently lost in cancers including breast, lung, colorectal, and ovarian cancers . ConsistOther E3 ubiquitin ligases including Skp2  and KLHLOTUD7B belongs to the ovarian tumor (OUT) family of deubiquitinases and prefers to hydrolyze the K63-linked ubiquitin chain . OTUD7B Despite the dramatic advance in understanding the roles and regulations of E3 ubiquitin ligases/DUBs in human diseases, only a few of their inhibitors are clinically used or in clinical trials . In the Since identification of the mTOR kinase in the 1990s, significant progress has been made in the mTOR research field, notably mTORC1 signaling. Many new components and regulators were discovered to make up the delicate mTOR network. Given the essential role of the mTOR pathway in many fundamental biological processes, complete loss of most mTOR components has been proved to be embryonically lethal in mouse, such as mTOR, Raptor, mLST8, Rictor, Sin1, Rheb, and RagA . TherefoTo date, more than 200 types of PTMs have been identified and play key roles in regulating protein functions in all signaling pathways. However, only a few kinds of PTMs have been validated in a dozen mTOR components and linked to the dysregulation of mTOR signaling. Therefore, identifying functional PTMs on mTOR components would be of great interest in future studies, which may provide more strategies for therapeutically targeting mTOR signaling in clinic.


Arabidopsis mutants. The discovery of the JA receptor, CORONATINE INSENSITIVE 1 (COI1), and the central repressors, jasmonate ZIM (JAZ)-domain proteins, further promotes the efforts to understand the JA signaling pathway in Arabidopsis. However, many aspects about the JA signaling pathway in other plant species remain to be elucidated.The plant hormone jasmonic acid (JA) and its derivative, an amino acid conjugate of JA (jasmonoyl isoleucine: JA-Ile), are signaling compounds involved in the regulation of cellular defense and development in plants. The number of articles on JA has increased dramatically since the 1990s. JA was recognized as a stress hormone that regulates plant responses to biotic stresses, such as those elicited by hervivores and pathogens, as well as abiotic stresses, such as wounding and ultraviolet radiation. Recent studies have progressed remarkably in understanding the importance of JA in the life cycle of plants. It has been revealed that JA is directly involved in many physiological processes, including stamen growth, senescence, and root growth. Furthermore, it has been known to regulate the production of various metabolites, such as phytoalexins and terpenoids. Many active regulatory proteins involved in the JA signaling pathway have been identified by screening for Arabidopsis: Camellia sinensis [Panax ginseng [Oryza sativa L. [Prunus avium L. [Brassica rapa [Sorghum bicolor L. [Nicotiana benthamiana [Pogostemon cablin [Zea mays [Arabidopsis thaliana [This Special Issue, “Jasmonic Acid Pathway in Plants” contains five review articles published by field experts. Information available on the important role of JA in plant growth has been updated in these reviews ,2,3,4,5.sinensis , Panax g ginseng , Oryza sativa L. ,9, Prunuavium L. , Brassicica rapa , Sorghumcolor L. ,13, Nicothamiana , Pogosten cablin , Zea mayZea mays , and Arathaliana ,18,19,20thaliana  and DampXanthomonas oryzae pv. oryzae (Xoo), in rice [Xoo resistance in rice [Xoo [Xoo. Taken together, these results strongly indicate that the JA and SA signaling pathways interact coordinately to yield induced defense responses in rice. Rice may develop a unique system to shield itself against pathogens. Recently, this unique system has been called “Common Defense System” [Uji et al. demonstrated that the JA-induced VQ-motif-containing protein, OsVQ13, positively regulates the JA signaling pathway in rice . Interes in rice . General in rice . JA-induice [Xoo ,23. Uji ice [Xoo . It has  System” .Ralstonia solanacearum is known to be hemibiotrophic and causes bacterial wilt disease in more than 200 plant species, such as tomato, potato, banana, and eggplant. Plants have developed a specialized defense system, the so-called pattern-triggered immunity (PTI), to inhibit or attenuate infection due to R. solanacearum. To suppress PTI, the pathogen injects approximately 70 type III effectors into the plant cells through the Hrp type III secretion system. Nakano and Mukaihara identified an effector, RipE1, which promoted the degradation of JAZ repressors and induced the expressions of JA-responsive genes in a cysteine–protease-activity-dependent manner [N. benthamiana. Thus, the activation of the JA signaling pathway causes the suppression of the SA signaling pathway, which is essential for the defense response against R. solanacearum. They also revealed that the effector RipAL induces JA production to activate its signaling pathway and simultaneously suppress the SA-mediated defense response in these plants [R. solanacearum hijacks the JA signaling pathway and exploits antagonistic interactions between the JA and SA signaling pathways to promote a successful infection. They unraveled one of the survival strategies, which was previously unknown.Nakano and Mukaihara published an excellent study in this Special Issue . The patt manner . JA and e plants . These rFinally, I would like to express my heartfelt gratitude to all of the authors and referees for their tremendous and relentless efforts in supporting this Special Issue. Without their valuable assistance, I would not have had an opportunity to publish this timely and successful publication, with its useful updates on the JA signaling pathway in plants. I also thank the assistant editor Sydney Tang for supporting my works.


Sarcopenia is characterized by a progressive skeletal muscle disorder that involves the loss of muscle mass and low muscle strength, which contributes to increased adverse outcomes. Few studies have investigated the association between chronic infection and sarcopenia. This study aimed to examine the association between human T-cell lymphotropic virus type-1 (HTLV-1) and sarcopenia. We conducted a cross-sectional study and enrolled 2,811 participants aged ≥ 40 years from a prospective cohort study in Japanese community dwellers during 2017–2019. Sarcopenia was defined as low appendicular skeletal muscle mass and low handgrip strength. The association between HTLV-1 seropositivity and sarcopenia was assessed using multivariable logistic regression. Odds ratio (OR) and 95% confidence interval (CI) of sarcopenia were analysed using HTLV-1 seropositivity. We adjusted for age, sex, body mass index, physical activity, systolic blood pressure, glycated haemoglobin, low-density lipoprotein cholesterol, and smoking and drinking status. Of 2,811 participants, 484 (17.2%) HTLV-1 infected participants were detected. HTLV-1 infection was significantly associated with sarcopenia . HTLV-1 was associated with sarcopenia among community-dwelling adults. Active surveillance and early detection of asymptomatic HTLV-1 infection might be beneficial to reinforce countermeasures to inhibit the progress of HTLV infection-associated sarcopenia. Sarcopenia is characterized by a progressive and generalized skeletal muscle disorder that involves the accelerated loss of muscle mass and low muscle strength . SarcopeFactors that cause and worsen sarcopenia are categorised as primary and secondary. Known risk factors include changes in sex steroid hormones and cytokines associated with aging, genetic susceptibility (as primary sarcopenia), physical inactivity, smoking, low 1,25-OH vitamin D, low nutritional intake, low protein intake, and atherosclerosis (as secondary sarcopenia) , 8–17.A recent study reported that sarcopenia was more prevalent in a community cohort of human immunodeficiency virus (HIV) infection . In the + T-lymphocytes were found to infiltrate the muscle of patients with inflammatory myopathies, and cytotoxic T-lymphocyte immune reactions to the TAX gene induced tumour necrosis factor-alpha and cytotoxic effects on muscles  ± 10.0), 502 (18%) HTLV-1-infected participants were detected using a chemiluminescent enzyme immunoassay (CLEIA). Samples from 488 of these 502 participants were available for real-time PCR (RT-PCR) testing. Of these 474 were positive for HTLV-1 and 14 were negative by RT-PCR. For the negative cases, western blotting showed that three cases were positive, one was negative, one was indeterminate, and Innogenetics line immunoassay showed that seven cases were positive, one was indeterminate, and one was negative. The 484 (17%) participants who were positive for HTLV-1 by RT-PCR, western blotting or Innogenetics line immunoassay were included in the analysis.In univariable linear regression analysis, HTLV-1 infection had a positive association with sarcopenia  . The staIn logistic regression analysis, HTLV-1 infection was significantly associated with low handgrip strength . This association remained significant after further adjustment for age, sex, BMI, physical activity, SBP, HbA1c, low-density lipoprotein cholesterol (LDL), and smoking and drinking status  . HTLV-1 We also tested the association between HTLV-1 infection and sarcopenia among participants who were excluded as premenopausal women to avoid pre/postmenopausal changes to muscle mass and strength. The aOR did not differ after excluding premenopausal women .Logistic regression analysis was performed to examine the association between sarcopenia and inflammatory markers (logarithm of hs-CRP and WBC). Hs-CRP was significantly associated with low SMI after adjustment for age, sex, BMI, physical activity, SBP, HbA1c, LDL, and smoking and drinking status  . WBC wasThis community-based cross-sectional study demonstrated that asymptomatic HTLV-1 infection is a risk factor for sarcopenia. To the best of our knowledge, this is the first report to show chronic HTLV-1 infection is a risk factor for sarcopenia. In addition, inflammatory makers were identified as risk factors for low SMI and sarcopenia. Sarcopenia has recently been recognized as an emerging issue in HIV infection . MulticeA putative mechanism of HTLV-1 associated sarcopenia is warranted. Potential explanations include anabolic resistance induced by inflammation and triggered catabolism. Because HTLV-1 promotes the production of cytokines such as interferon-gamma, tumour necrosis factor-alpha, and interleukin-6 , throughTAX, which increases the production of ROS [+ transport mechanism altered [Another putative pathway of HTLV-1 associated sarcopenia is an overabundance of reactive oxygen species (ROS). HTLV-1 encodes viral structural genes of the pX region encoding n of ROS , 30. ROSn of ROS . ROS con altered .In a retrospective study, thirteen French HTLV-1 infected-patients with polymyositis or dermatomyositis showed moderate muscle inflammation compared with HTLV-1 infected-controls without myopathy. However, the level of the proviral load did not differ between these groups . AlthougThe associations between HTLV-1 infection and low handgrip strength, and HTLV-1 infection and sarcopenia were significant, but not between HTLV-1 infection and low SMI. These inconsistent results may be partially explained by the loss of muscle strength, which is typically clinically apparent before the loss of muscle mass. Data from a longitudinal study in Baltimore showed that a decline in muscle strength was much greater than that predicted by the decline in muscle mass .Some limitations of the present study should be mentioned. First, as this was a cross-sectional study, we were not able to establish cause–effect relationships. Second, we had no data on the infectious period of each participant. Because effects of HTLV-1 infection on sarcopenia progression are thought to be cumulative, differences in the duration of infection may bias the results. Third, although the study participants were recruited from healthy community dwellers, we cannot deny the mixture of HTLV-1 infected-patients with polymyositis or dermatomyositis. However, although the association between HTLV-1 infection and sarcopenia was not significant, the same tendency was observed after excluding participants with joint pain (n = 639), which is a common symptom of inflammatory myopathy  .In conclusion, HTLV-1 was associated with sarcopenia in Japanese community dwellers. Similar to HIV infection, active surveillance and early detection of asymptomatic HTLV-1 infection might help reinforce countermeasures to prevent and inhibit the progress of HTLV-1 infection associated sarcopenia.We conducted this cross-sectional study using data from the Nagasaki Islands Study, which was a prospective cohort study performed in Goto City in the western islands of Japan . DetailsThe participants were recruited at medical check-ups, and members of the general population aged ≥ 40 years living in Goto City were targeted for enrolment. The Ethical Committee of Nagasaki University approved this study , which was conducted according to the ethical standards defined in the 1964 Declaration of Helsinki as well as its subsequent amendments. Written informed consent was obtained from all participants. Of 3,365 participants enrolled from 2017 to 2019, 2,811 participants were included after exclusion for the following: age <40 years (n = 73), history of stroke (n = 155), BMI < 18.5 to avoid the effects of undernutrition on sarcopenia (n = 233), missing data for HTLV-1 serostatus (n = 14), handgrip strength (n = 7), ASM (n = 70), LDL (n = 1), and history of hypoglycaemic agent use (n = 1). Finally, 2,811 participants  with a mean age of 70.2 years  were evaluated.2) was calculated as the ASM (kg) divided by the square of the height (m) defined by Baumgartner’s formula [Body weight and height were measured with participants wearing light-weight clothes and without shoes, and the BMI was then calculated. Body composition was analysed by multi-frequency bioelectrical impedance analysis using InBody 430 . The muscle mass of the four limbs as ASM was summated. SMI (kg/m formula . HandgriSBP and diastolic blood pressure (DBP) at rest were recorded with a blood pressure measuring device . The measurements were repeated when the SBP was ≥ 140 mmHg or DBP was ≥ 90 mmHg, and the mean values were used for analyses. We used a questionnaire to obtain information regarding each participant’s medical history of stroke, use of antihypertensive agents, hypoglycaemic agents, and lipid-lowering drugs. Smoking status and drinking status were categorized as “never”, “ex”, or “current”. Physical activity was evaluated using two questions: “Do you have a daily walking habit?” and “Do you regularly exercise more than 30 minutes in a day? ”. Physical activity was scored as the total sum of “Yes” answers to the two questions.Fasting blood samples were collected at the time of clinical examination. Blood samples were collected in an EDTA-2K tube, a siliconized tube, and a sodium fluoride tube. Samples from the EDTA-2K tube were used to measure the levels of WBC using an automated procedure at SRL, Inc. . Serum concentrations of LDL, HDL, TG, creatinine, HbA1c, and hs-CRP were measured by standard laboratory procedures.CLEIA kit  was used for HTLV-1 detection. As a confirmatory test, we used RT-PCR using the Hydrolysis probe method with the LightCycler 480  as previously reported . Sample 2 for men and <5.7 kg/m2 for women were diagnosed as sarcopenia using Asian cutoff values [Patients with a handgrip strength <26 kg for men and <18 kg for women, and SMI <7.0 kg/mf values . Low hana priori for age, BMI, SBP, HbA1c, LDL, physical activity (continuous variables), sex (dichotomous variable), and smoking and drinking status  [Differences in mean values or proportions of variables by HTLV-1 positivity were analysed using the Wilcoxon rank sum test for continuous variables , or the McNemar chi-square test for categorical variables . We performed simple linear regression analysis of handgrip strength and ASM as continuous variables. Then, we performed logistic regression analysis using clinical cutoff points of low handgrip strength, low SMI and sarcopenia. In the multivariable logistic regression analysis, adjustments were made ctively) , 35. AgeBecause markers of chronic inflammation may play an important role in sarcopenia progression , we also


Cell therapies represent a promising approach to slow down the progression of currently untreatable neurodegenerative diseases , as well as to support the reconstruction of functional neural circuits after spinal cord injuries. In such therapies, the grafted cells could either functionally integrate into the damaged tissue, partially replacing dead or damaged cells, modulate inflammatory reaction, reduce tissue damage, or support neuronal survival by secretion of cytokines, growth, and trophic factors. Comprehensive characterization of cells and their proliferative potential, differentiation status, and population purity before transplantation is crucial to preventing safety risks, e.g., a tumorous growth due to the proliferation of undifferentiated stem cells. We characterized changes in the proteome and secretome of human neural stem cells (NSCs) during their spontaneous  differentiation and differentiation with trophic support by BDNF/GDNF supplementation. We used LC-MS/MS in SWATH-MS mode for global cellular proteome profiling and quantified almost three thousand cellular proteins. Our analysis identified substantial protein differences in the early stages of NSC differentiation with more than a third of all the proteins regulated  and revealed that the BDNF/GDNF support affected more the later stages of the NSC differentiation. Among the pathways identified as activated during both spontaneous and BDNF/GDNF differentiation were the HIF-1 signaling pathway, Wnt signaling pathway, and VEGF signaling pathway. Our follow-up secretome analysis using Luminex multiplex immunoassay revealed significant changes in the secretion of VEGF and IL-6 during NSC differentiation. Our results further demonstrated an increased expression of neuropilin-1 as well as catenin β-1, both known to participate in the regulation of VEGF signaling, and showed that VEGF-A isoform 121 (VEGF121), in particular, induces proliferation and supports survival of differentiating cells. PCNA pin vitro, which are independent on oxygen level.Among the pathways identified as activated during both S and BG differentiations in our SWATH-MS data were the HIF-1 signaling pathway, Wnt signaling pathway, and VEGF signaling pathway. HIF1-α activates gene expression in response to hypoxia including gene for VEGF  than VEGF165 may play a neuroprotective role  at the early stage of NSC differentiation, which can be assigned to a change of the microenvironment, but activation of Caspase-3 was decreased at a later stage of both differentiations, which may correlate with increased secretion of VEGF-A.in vitro and in vivo  studies related to CNS pathologies, including autism spectrum disorders , and by the fluctuating levels of the apoptotic marker caspase-3. H9 NSCs on their own secreted increasing levels of IL-6 and VEGF-A over the differentiation time course. This secretory phenotype could be potentially beneficial as part of neuroprotective and modulatory effect of transplanted cells in cell-based therapies.Overall, our results show large-scale proteome changes in human H9 NSCs differentiating https://panoramaweb.org/NSCsdifferentiation.url) and on ProteomeXchange under ID PXD021860.The datasets from the SWATH-MS measurements for this study can be found in the Panorama Public repository (JČ, JT, PV, and HK designed the study. JČ, JT, HK, KV, IP, IV, TP, and PV performed the experiments. JČ, JT, KV, HK, LP, MK, MV, and PV analyzed and interpreted the data. JČ, JT, HK, and PV wrote the paper with input from all authors. JČ, HK, and PV received the funding. All authors have seen and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Skeletal dysplasias are a diverse group of heritable diseases affecting bone and cartilage growth. Throughout the years, the molecular defect underlying many of the diseases has been identified. These identifications led to novel insights in the mechanisms regulating bone and cartilage growth and homeostasis. One of the pathways that is clearly important during skeletal development and bone homeostasis is the Wingless and int-1 (WNT) signaling pathway. So far, three different WNT signaling pathways have been described, which are all activated by binding of the WNT ligands to the Frizzled (FZD) receptors. In this review, we discuss the skeletal disorders that are included in the latest nosology of skeletal disorders and that are caused by genetic defects involving the WNT signaling pathway. The number of skeletal disorders caused by defects in WNT signaling genes and the clinical phenotype associated with these disorders illustrate the importance of the WNT signaling pathway during skeletal development as well as later on in life to maintain bone mass. The knowledge gained through the identification of the genes underlying these monogenic conditions is used for the identification of novel therapeutic targets. For example, the genes underlying disorders with altered bone mass are all involved in the canonical WNT signaling pathway. Consequently, targeting this pathway is one of the major strategies to increase bone mass in patients with osteoporosis. In addition to increasing the insights in the pathways regulating skeletal development and bone homeostasis, knowledge of rare skeletal dysplasias can also be used to predict possible adverse effects of these novel drug targets. Therefore, this review gives an overview of the skeletal and extra-skeletal phenotype of the different skeletal disorders linked to the WNT signaling pathway. In the last decades, plenty of examples have been generated where the identification of causal genes for rare, monogenic diseases resulted in novel insights into the role and functioning of already known pathways. A textbook example of this is Wingless and int-1  signaling and its role in bone formation and homeostasis. First indications of this involvement were generated by studying rare monogenic conditions with abnormal bone mass, delivering novel targets for drug development for osteoporosis, a common disorder characterized by decreased bone mass in late life. Recently, an antibody against a WNT signaling inhibitor was approved as an anabolic agent for the treatment of osteoporosis.2+-dependent pathway and the WNT/planar cell polarity (PCP) pathway  which was the first member from this gene family being described  which wa. Meanwhi pathway . All thrIn mammalians, 19 different WNT ligands have been identified and 10 FZD receptors , alreadyAt the beginning of this century, an additional role of WNT signaling was discovered following new gene identifications in some rare monogenic skeletal dysplasias. As explained in detail below, the study of conditions with either decreased or increased bone mass resulted in the identification of mutations in several genes involved in especially canonical WNT signaling . The mosAs previously mentioned, the WNT signaling pathway is activated by the binding of WNT ligands. Because of the broad functions of this pathway, additional regulation mechanisms are required to ensure proper timely and spatially functioning of the pathway. Extracellular modulators, including WNT inhibitors and activators, contribute to this complex regulation. Not surprisingly, mutations in various components of this pathway have been described in skeletal dysplasias.WNT ligands are secreted glycoproteins with a length of 350–400 amino acids. In humans, 19 different ligands have been identified, all containing 23–24 conserved cysteine residues , 8. A diWNT1 gene are found in families with osteogenesis imperfecta (OI) type XV and early-onset osteoporosis  in the WNT3 gene has been reported in tetra-amelia syndrome type 1  type 1 (ADRS1)  1/2 –24. Hete (ADRS1) . RS is cWNT6 gene is located. As this gene is expressed during limb development, it is a promising candidate gene for harboring disease-causing mutations for F-syndrome  is a rare skeletal disorder with a dominant inheritance pattern , 29. SymWNT7A cause limb malformations in Al-Awadi–Raas–Rothschild syndrome (AARRS) and Fuhrmann syndrome (FS)  –37. Patiome (FS) –37. Althome (FS) , 38, 39.The WNT10B ligand is a key regulator for osteogenesis and is involved in all stages of limb development , 40–42. SOST gene, is an osteocyte-derived secreted glycoprotein with a cysteine-knot motif  inhibitor that regulates limb and kidney formation during development . Howevermalities , 62–64. s  4 is one of the five members of the sFRP family, known for antagonizing the WNT signaling pathway –70. sFRP disease , 73. The disease , 73.via a glycosyl-phosphatidylinositol (GPI) anchor , a subfamily of heparin sulfate proteoglycans, are linked to the cell surface ) anchor –77. The ) anchor . Six higGPC4 is located on chromosome Xq26 and is a regulator of the WNT/PCP pathway , 81. OnlGPC6 prevent anchoring to the membrane, thereby disrupting the action of GPC6 in developmental processes (FZD2 gene (see below).GPC6 is the smallest protein of the GPC family and is widely expressed in the growth plate during skeletal development , 77, 85.rocesses –77. Suchrocesses . This rarocesses , 77. TheWNT3 alterations (see above), an impaired RSPO2 function contributes to the development of tetra-amelia. However, patients affected with RSPO2 mutations exhibit additional hypoplasia or aplasia of the lungs (R-spondins (RSPO) are secreted proteins and activators of the canonical WNT signaling pathway –88. The he lungs . Bi-allehe lungs . Furtherhe lungs .Both the canonical and non-canonical WNT signaling pathways are activated by the binding of WNT ligands to the FZD receptors alone or in combination with specific co-receptors. Identification of single disease mutations in LRP5, LRP6, and ROR2 highlights the importance of these co-receptors in the regulation of canonical and non-canonical WNT signaling during skeletal development.FZD2 can cause autosomal dominant OMOD, also referred to as OMOD2. OMOD2 is a rare skeletal dysplasia which is marked by severe rhizomelic shortening of the upper limbs, mild facial dysmorphism , and genital hypoplasia (FZD2 is a member of the FZD receptor family that includes nine G protein-coupled receptors. These FZD receptors interact with disheveled (DVL) upon activation by one of the WNT ligands which results in the initiation of one of the different WNT pathways. In 2015, Saal et al.  demonstrpoplasia . Clinicapoplasia .FZD2 can cause OMOD2. To date, one missense mutation  and two nonsense mutations involving adjacent amino acids (p.Ser547* and p.Trp548*) have been reported  or autosomal recessive RS type 1 (ARRS1) , and a Kringle domain (KD). The intracellular region contains a tyrosine kinase (TK) domain followed by serine/threonine-rich and proline-rich structures. Mutations in  (ARRS1) , 97.ROR2 as a disease-causing gene for ARRS1, more than 25 different mutations have been identified . In addition, patients with ARRS1 may also have vertebral segmentation defects such as hemivertebrae and rib fusions . Since tentified . These mentified , 98–101. protein .ROR2 are all nonsense or frameshift mutations that are located in the intracellular region of the protein and co-localize in two domains, either immediately N-terminal or C-terminal of the TK domain  phenotype, autosomal dominant osteopetrosis type 1, autosomal dominant Van Buchem disease, Worth disease, endosteal hyperostosis, or osteosclerosis. Although these rare disorders have a different name, they represent the same disorder which is characterized by increased bone mass especially affecting the skull and tubular bones and a reduced fracture risk , isolated syndactyly, and congenital myasthenia gravis depending on type and location of the mutations  which all act downstream of FZD receptors in both the canonical and non-canonical WNT signaling pathway. The DVL proteins share highly conserved regions, namely, the amino-terminal DIX (N-terminus) domain, a PDZ  domain, a carboxyl-terminal DEP (C-terminus) domain, and two regions with positively charged amino acid residues . These cDVL1 and DVL3 have been identified in individuals with autosomal dominant RS (ADRS) types 2 and 3, respectively   , 130.DVL1 but also DVL3 can cause ADRS (DVL3 (ADRS3) can be distinguished from ADRS2 patients based on the presence of short stature and macrocephaly. Furthermore, congenital heart defects and cleft lip and/or cleft palate were reported in the majority of the patients . In two families without mutations in the known causative gene ROR2, White et al. (Nucleoredoxin (NXN) is an oxidative stress response protein which is highly expressed during murine limb bud development and can directly bind DVL pathways –136. It pathways . Recentle et al.  identifie et al. .Figure 3), they suggest that the identified de novo variant can cause the Robinow-like phenotype in the patient /c-jun phosphorylation which is necessary for cytoskeletal organization. White et al.  reported patient . However patient  also ideAdenomatous polyposis coli (APC) is a tumor suppressor gene that can regulate canonical WNT signaling as a part of the multiprotein destruction complex that targets β-catenin for phosphorylation and degradation  (APC Membrane Recruitment Protein 1 (AMER1), also known as FAM123B or Wilms tumor on the X chromosome (WTX), is an intracellular inhibitor of the canonical WNT signaling pathway. AMER1 can interact with the AXIN/APC/GSK3β/β-catenin multiprotein complex and enhances the ubiquitination and degradation of β-catenin , 144. AMs (OSCS) . Radiogrs (OSCS) , 146.PORCN is located on the X-chromosome, and heterozygous loss-of-function mutations can cause focal dermal hypoplasia (FDH) also known as Goltz syndrome in female patients. Goltz syndrome caused by mutations in PORCN is rarely reported in males which leads to the suspicion that non-mosaic Goltz syndrome is embryonically lethal for males (Porcupine O-acyltransferase (PORCN) is a member of the evolutionarily conserved porcupine (PORC) gene family of endoplasmic reticulum transmembrane proteins that are involved in the processing and secretion of WNT ligands. or males , 148. Goor males .SOST, LRP4, LRP5, and LRP6, are all involved in the canonical WNT signaling pathway , a first-line treatment for bipolar disorders, increases canonical WNT signaling activity and increases bone formation already confirms the therapeutic potential of the pathway , 159. Bur et al. , and shor et al. . Also focer risk –166. HowCurrently, the treatment of osteoporosis is mainly based on the use of antiresorptive agents, such as bisphosphonates, without the capacity to rebuild the lost bone. Consequently, there is an urgent need for more specific, anabolic therapies for osteoporosis, making canonical WNT signaling of major interest. The pharmaceutical industry focused on sclerostin as a therapeutic target based on the expression of sclerostin being almost exclusive in bone tissue, the progressive increase in bone mass in adults with sclerosteosis and Van Buchem disease and on the absence of non-skeletal clinical features in these patients . ClinicaIn conclusion, mutations in the WNT signaling pathway can lead to a broad range of skeletal dysplasias. Especially those characterized by an increased bone mass have been instrumental in highlighting the role of WNT signaling in bone formation and homeostasis with therapeutic applications for the treatment of osteoporosis.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Human T-cell lymphotropic virus type 1 (HTLV-1) infects 5–10 million people worldwide and is the causative agent of adult T-cell leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) as well as other inflammatory diseases. A major concern is that the most majority of individuals with HTLV-1 are asymptomatic carriers and that there is limited global attention by health care officials, setting up potential conditions for increased viral spread. HTLV-1 transmission occurs primarily through sexual intercourse, blood transfusion, intravenous drug usage, and breast feeding. Currently, there is no cure for HTLV-1 infection and only limited treatment options exist, such as class I interferons (IFN) and Zidovudine (AZT), with poor prognosis. Recently, small membrane-bound structures, known as extracellular vesicles (EVs), have received increased attention due to their potential to carry viral cargo (RNA and proteins) in multiple pathogenic infections , Zika virus, and HTLV-1). In the case of HTLV-1, EVs isolated from the peripheral blood and cerebral spinal fluid (CSF) of HAM/TSP patients contained the viral transactivator protein Tax. Additionally, EVs derived from HTLV-1-infected cells (HTLV-1 EVs) promote functional effects such as cell aggregation which enhance viral spread. In this review, we present current knowledge surrounding EVs and their potential role as immune-modulating agents in cancer and other infectious diseases such as HTLV-1 and HIV-1. We discuss various features of EVs that make them prime targets for possible vehicles of future diagnostics and therapies. Strongyloides stercoralis, and Norwegian scabies . Th. Th+CD25Finally, there is very little information on whether Tax within EVs could be involved in the induction of proinflammatory cytokines in recipient CNS cells ; and whether HTLV-1 EVs containing cytokines play a role in HTLV-1-associated HAM/TSP. Moreover, it is not clear whether HTLV-1 EVs could participate in the spontaneous lymphoproliferation observed in HAM/TSP patients. To better understand the comprehensive mechanism of EV-mediated inflammation in CNS, a detailed study oriented towards EVs and HAM/TSP performed from multiple patient sample cohorts could address these potentially significant issues. + and CD8+, a CD4+/CD8+ ratio of 1:73, and hyperexpression of IgD or IgE [+ T lymphocytes in IDH patients have not been found to have cytotoxic effect  [Staphylococcus aureus (S. aureus) and beta-haemolytic streptococci (BHS) [+ T cells) and tumorigenesis [Infective dermatitis associated with HTLV-1 (IDH) is a rare but important HTLV-1-related disease due to increases in the geographical distribution ranging from the Caribbean  to South America  and Africa  . IDH is D or IgE ,163. Moserforin) , and theci (BHS) . The mecci (BHS) . Moreoveigenesis . Additioigenesis ,170. TheS. aureus and/or BHS [S. aureus induced keratinocyte death, skin barrier disruption and inflammation —the key phenotypes associated with atopic dermatitis (AD) [S. aureus EVs [S. aureus EVs [S. aureus EVs mediate pathogenesis of AD and could be used as a therapeutic target to reduce AD spread. However, the role of EVs in infective dermatitis largely remains unexplored. A study showed that 5 out of 35 patients with AD and all 50 patients with IDH had been diagnosed with HTLV-1, and both groups had d/or BHS . Anothertis (AD) ,173,174.reus EVs . Additioreus EVs . This wareus EVs . AltogetA recent study performed in a mouse model showed that adipose tissue-derived mesenchymal stem cell-derived exosomes (ASC exosomes) successfully decreased systemic inflammation and ameliorated AD symptoms . Ceramid+ T cells. Elucidating the mechanism underlying how HTLV-1 EV-associated cytokines (whether surface-bound or encapsulated) mediate the mechanism of CD8+ T cell inactivation and IDH pathogenesis could reveal possible mechanisms involved in IDH progression.In addition to the aforementioned works on IDH and AD, several studies have shown the role of EVs in HTLV-1 pathogenesis. EVs isolated from cell lines, PBMCs, and CSF samples have been shown to contain Tax protein. Although not proven, but HTLV-1 EVs  could affect IDH pathogenesis by inactivating the cytotoxic functions of CD85 peripheral blood leukocytes), virus subtype , genetics, and limited cytotoxic T lymphocyte effect were linked to the severity of lung disease [+CD25+ T lymphocytes, release of cytokines , inflammatory chemokines , and expression of ICAM-1 in the bronchioalveolar lavage fluid (BALF) [FoxP3 mRNA in the BALF of patients with HTLV-1-related lung diseases [Tax and HBZ) in the BALF cells of patients with HTLV-1 [A recent case-controlled study performed in central Australia showed that bronchiectasis played a role in an increase in the death rate among HTLV-1-positive Indigenous Australian adults . High vi disease . High-re disease . A serie disease ,181,182  disease ,184,185.d (BALF) ,186. Thediseases . Moreoveh HTLV-1 . Tax is h HTLV-1 . Taken tRegarding the role of EVs in pulmonary disease, studies have shown the role of EVs in other pulmonary diseases such as chronic obstructive pulmonary disease (COPD), pulmonary hypertension (PH), and Asthma, among others. A series of studies have shown the role of specific miRNA containing exosomes in pulmonary diseases such as increased concentration of miR-210  and EV-m+ T cells that stimulate the release of inflammatory cytokines  [+ T lymphocytes, CD4/8 ratio, CD25+ T lymphocytes (express IL-2R), as well as decreased CD8+ T lymphocytes were observed in HU patients [HTLV-1-associated uveitis (HU) is one of the most common uveitis in endemic areas of Japan . HU is a GM-CSF) ,199, res GM-CSF) . Topical GM-CSF) ,199. The GM-CSF)  that can GM-CSF) . Additiopatients , pointinAdditionally, HTLV-1 negatively affects the retinal pigment epithelium (RPE) of the eye which is a monolayer of cells located between the neuroretina and the vascularized choroid. RPE is crucial in maintaining the homeostasis of the outer retina which is considered part of the blood-ocular -barrier (BOB) that protects the eye. RPE expresses various cytokine receptors and their ligands including IL-1, IL-6, IL- 15, TNF, TGF, FGF, IGF, VEGF and PDGF, major histocompatibility complex (MHC) molecules, cell surface adhesion molecules , and Fas receptor-Fas ligand (FasL) that are involved in immune and inflammatory responses . ICAM-1 +CD16+) as well as the release of TGF-β1 [Even though various studies have explored the pathogenesis of HU, investigating the role of HTLV-1 EVs in HU could potentially paint a clearer picture of the disease and may even provide new therapeutic venues. The role of EVs in regulating immune responses in non-infectious uveitis patients  have been investigated in few studies. Human PBMCs from non-infectious uveitis patients exposed to EVs isolated from both unstimulated and cytokines -stimulated RPE cells have been found to block T cell proliferation with no reduction in T cell viability . Furtherf TGF-β1 . These ff TGF-β1 , thus blAdditionally, an increase in EV concentration was observed after RPE cells were exposed to an oxidative stressor . These E+ T cells (main cell targets for HTLV-1 infection) in patients with HTLV-1 has also been studied [+ T cells [+ T cells [Currently, there are no FDA-approved drugs for the treatment of HTLV-1 infection. However, associated disorders can be managed, and antiviral drugs are being repurposed and developed. Reverse transcriptase (RT) inhibitors such as Zidovudine (AZT) and phosphonated carbocyclic 2′-oxa-3′aza nucleosides (PCOANs) have demonstrated consistent inhibition of RT from HTLV-1 isolated from the patients . Other p studied . The ant studied . An impr studied . Overall T cells . Lastly, T cells ,216, sug T cells . FinallyCurrently there are no widely accepted inhibitors of EV biogenesis, release (secretion), or uptake, however identification of such drugs may provide valuable options to prevent disease progression. For instance, EV secretion is an important feature of many neoplastic disorders, including ATLL. EVs facilitate tumor growth, immune evasion, tumor angiogenesis, metastasis, and resistance to therapeutic agents by spreading tumor growth factors and modulating immunological tolerance . Tumor-dCurrent EVs inhibitors can be classified into three categories based on their pharmacological effect on inhibiting EV trafficking (secretion), lipid metabolism (biogenesis), or uptake. Calpeptin, MDL28170, Manumycin A, Tipifarnib, Y-27632, and Neticonazole are drugs known to target EV trafficking, while GW4869, Imipramine, and Pantethine impact the lipid metabolism of molecules that are involved in EV production and secretion ,220,221.+) levels and cytoskeletal remodeling [+-dependent cytosolic cysteine protease. Mechanistically, this drug interrupts the binding of calpain and cortactin (an actin-nucleation factor), restricting MVB formation and protein packaging into vesicles. These events may result in a reduction of EV release  [I to activate the surface protein LFA-1 in T-cells, promoting cell-cell contact and viral spread [+-dependent signaling. HTLV-1 infection via cell-cell contact was downregulated after calpeptin treatment [It has been observed that cellular activation may change the levels of EVs trafficked and secreted by a cell, modifying the functional effects on recipient cells. EVs may be necessary to maintain homeostasis. For instance, cellular activation has been shown to yield higher levels of EV secretion compared to resting cells. This upregulation in EV trafficking has been observed to occur in parallel with increases in intracellular calcium  . HTLV-1 l spread . Calpeptreatment . Anotherreatment . Furtherreatment . Manumycreatment ,233. Botreatment ,233. Thereatment . These dreatment . Similarreatment . TipifarAdditionally, Y-27632 is an inhibitor of Rho-associated, coiled coil-containing protein kinase (ROCK). Y-27632 competes with ATP for binding to the kinase which hinders both ROCK1 and ROCK2 enzymes. ROCK induces actin reorganization via membrane deformation, which helps in vesicle formation. Y-27632 blocks EVs shedding from the cell membrane . NeticonEV release can also be inhibited by impairing lipid metabolism. The enzyme nSMase 2 induces the conversion of sphingomyelin to ceramide that is responsible for exosome biogenesis  and the shedding of MV from cells ,242. InhImipramine, in combination with other drugs, has been used as an antidepressant and neurogenic bladder therapy for HAM/TSP patients ,166. ImiDue to the heterogeneity of EV subpopulations , the inhibition of different pathways for EV biogenesis and release may be achieved by using a combination of some of the previously described inhibitors. Knowing which EV subpopulation is mainly responsible for disease development would be significant in pinpointing which of the EV type should be blocked from release. In this case, cell viability and proliferation should be measured to confirm that changes in EV quantities are related to the action of an inhibitor, and not to drug toxicity. Since EVs may play a role in the viral life cycle and in the associated disease progression, inhibition of EV release becomes an attractive option. The arsenal of EV release inhibitors is increasing over time and there are more drugs discovered to inhibit this process. EVs have numerous surface molecules such as proteins and glycoproteins that may bind many cell receptors, making possible the interaction between cells and exchange of cargo . Normal Various EV subpopulations may use multiple routes of entry into target recipient cells and may differ depending on the cell type. In regard to HTLV-1 EVs, the molecular mechanisms of uptake by recipient cells has not been extensively studied. EV dose, incubation time, pH, and temperature (37 °C vs. 4 °C) are factors that could be evaluated in order to shed light on the methods of EV uptake. Since EVs have been shown to mediate disease progression, preventing their uptake into recipient cells using different drugs or compounds could be a beneficial strategy. For instance, Dynasore is an inhibitor of Dynamin-2 that regulates EV uptake via clathrin- and caveolin-dependent endocytosis . It has There are many challenges of EV inhibitor-based therapeutics mainly due to the lack of understanding of EV biogenesis, and lack of in vivo studies. The optimal strategy to prevent the functional effects of an EV is to stop its biogenesis. However, we do not fully understand how to efficiently isolate and identify individual populations of EVs generated by the multiple EV biogenesis pathways . This is a current challenge faced by the entire EV field, where EV-associated proteins are being used as potential biomarkers to determine their origin. In HTLV-1, the overarching issues related to pharmacological inhibition of EVs are lack of understanding of the EV subpopulation that is targeted by a drug, the side effects, and the EV type responsible for pathogenesis. For example, the use of an EV inhibitor may have effects on certain EV subpopulations, while leaving others functional. It is important to first identify which EV type is being inhibited by a particular drug prior to use in EV therapeutic strategies. Additionally, the impact of these EV inhibitors on EV biogenesis/secretion from normal cells should be meticulously investigated. Distinguishing between non-pathogenic and potentially pathogenic EVs (derived from infected cells) based on specific cargo  would be challenging. However, identifying these EV populations would offer significant benefits for controlling disease progression. A combination of multiple drug types may be necessary to fully inhibit EV production. Albeit the complete shutdown of EV production could also result in undesired side effects related to deregulation of processes that depend on background levels of EV production. For instance, some side effects of the EV release inhibitor, Imipramine, may include blood disorders, suppression of the immune response, nausea, vomiting, and other complications . OverallEffective methods to control the transmission of HTLV-1 infection should be carefully considered in highly epidemic areas. In relapse cases, treatment options for HTLV-1-associated diseases are not as effective. Moreover, there is limited education and awareness of HTLV-1 and no available vaccinations, thereby making transmission one of the most significant contributors of the high prevalence observed worldwide. To tackle these issues, we must first develop effective modes of detecting the virus versus EVs and follow up with preventive and supportive strategies for the populations at risk. Most of nucleic acid or protein-based detection methods may typically analyze viral proteins and particles to diagnose and decide on a treatment course. However, the EVs in these samples may be informative and equally important. Current blood collection and techniques used to separate coagulation factors away from the plasma and subsequently isolate white blood cells discard the supernatant material that could be clinically relevant in the near future. Therefore, it is possible that the plasma used for diagnostic of HTLV-1 infection also carries EVs. Recent evidence has shown that the supernatant from HTLV-1-infected cells contains a mixture of EVs  and potential non-infectious viral particles . EV subtTo detect and identify regions of the world with increasing trends of transmission, universal screening requirements are necessary, especially in cases of blood donations/transfusions, prenatal care, emergency medicine, drug abuse, and during routine screening for sexually transmitted infections (STIs). These are the high-risk groups that ought to be targeted in order to curb further spread of the virus and distinguish between infections virions and EVs. Few countries including the United States, Canada, Western European countries , and Japan have applied routine screenings for HTLV-1 among blood donors . UnfortuEven though it was discovered over 30 years ago and despite its association with debilitating diseases, namely ATLL and HAM/TSP, HTLV-1 has remained relatively neglected by public health officials. Although approximately 5–10 million people are believed to be infected with HTLV-1, its global prevalence may be much higher than is currently estimated ,3,4,5. TThe current knowledge surrounding EV-associated functions, especially in the context of cancer and HIV-1, could also be applicable to HTLV-1 EVs. Along these lines, future research should focus on the effects of HTLV-1 EVs on cellular transformation. Studies of this nature could provide valuable insight into the mechanisms contributing to viral spread, modulation of the host immune response, and disease progression. For example, in certain cancers, it has been shown that the EV-associated integrin αvβ3 can reprogram recipient cells and promote a malignant phenotype ,249. MorFuture experiments should also aim to better define the biochemical and functional characteristics of different HTLV-1 EV subtypes. This information could be useful for determining whether certain subpopulations of EVs from HTLV-1-infected cells may be more toxic than others. To illustrate this point, a comparative analysis of the pathological proteins associated with EVs from amyotrophic lateral sclerosis (ALS) patients found that superoxide dismutase 1 (SOD1) was more enriched in exosomes, while TAR DNA-binding protein 43 (TDP-43) and the RNA-binding protein Fused in Sarcoma (FUS) were more concentrated in microvesicles. Interestingly, this same study found that the mean size of both exosomes and microvesicles from ALS patients was larger than those from healthy controls . Therefo


Hot-band absorption and anti-Stokes emission properties of an organic fluorescent dye, Alexa Fluor 568, were characterized and compared with those of Rhodamine 101. The comparison of the properties  between the two dyes confirms that both dyes undergo the same process when excited in the red spectral region. Possible undesirable crosstalk effects and applications in dSTORM microscopy were demonstrated and discussed. S1 or S2 electronic states from the S0 ground state. Due to vibrations, the electronic states are broadened; after the photon absorption step, the molecule rapidly relaxes to its lowest vibrational level of S1 (Kasha’s rule) [S0 state and emits a single fluorescence photon. Because of the energy loss due to internal conversion, the emitted photon has a longer wavelength, which is called the Stokes shift [The emission spectra of fluorescent dyes can be elucidated via radiative/non-radiative transitions between the electronic states and internal conversions. Typically, the process begins with a single photon absorption when the molecule is excited to its higher S0 and S1 to excite the molecule. This is a nonlinear third-order process that was originally predicted in 1931 [However, during the anti-Stokes fluorescence, the emitted photon has higher energy (lower wavelength) than the excitation photon. This process requires some additional energy. The thermodynamic background of this process was highly debated in 1946 by Pringsheim and Vavilov and was eventually solved by Landau in the same year . Several in 1931 . BecauseT1 level is used as an intermediate energy storage state. During this process, two excited molecules undergo energy transfer. One returns to its ground S0 state while the other one excites to the S1 state. As a result, the emission spectrum exhibits fluorescence characteristics. This process is also called P-type delayed fluorescence [The triplet-triplet annihilation is a similar process, when the triplet rescence , 7.T1 state are considered. Because of the long lifetime of the triplet state, there is a possibility for the molecule to cross into the S1 state via temperature activation (reverse intersystem crossing). During the relaxation, emission can occur from the S1 or T1 states depending on whether the reverse intersystem crossing occurred. Because it was first observed in eosin [There are two single photon excitation models that produce anti-Stokes fluorescence. In the first model, molecules that are excited to the in eosin  and the S0 to S1. Because at thermal equilibrium the occupation of the vibrational levels follows the Boltzmann distribution, the possibility of absorption from higher energy levels is low and temperature-dependent. However, the lifetime and emission spectra are not affected. The hot-band absorption has been observed with several organic dyes: Rhodamine 101 (Rh101) [In the second single-photon model, the molecule is excited from a higher vibrational state of  Rh101) , Rh6G [11 , Rh6G  (Rh101) , Oxazine (Rh101) , and cya (Rh101) . Because (Rh101) , 15–18. Assuming a two-channel two-colour super-resolution dSTORM imaging assay with a sample simultaneously labelled with AF568 and AF647 dyes Fig. , the int− 4 M solution of methanol and PBS (1:1). Because of the low emission intensity, an additional bandpass emission filter (Semrock 582/75) was used after the main dichroic mirror (Semrock Di01-R405/488/532/635) and quad-band bandpass emission filter (Semrock FF01-446/510/581/703). For each laser setting, a single frame was captured with an exposure time of 100 ms using an EMCCD camera (Andor iXon 897). The lasers were fibre coupled, collimated, and expanded before they were focused into the back focal plane of the microscope objective. Assuming a Gaussian beam shape in the image plane, two Gaussian distribution curves were fitted on the captured images along each axis. Using the fitted parameters, the volume under the two-dimensional Gaussian distribution was calculated, and the background noise was subtracted. This value was used to indicate the intensity of the anti-Stokes fluorescence.First, we determined the function between the excitation power and emitted intensity at different excitation wavelengths . The output power of each source was calibrated and measured after the microscope objective (Nikon CFI Plan Apo Lambda 60x oil). Both Rh101 and AF568 dyes were prepared in a 10Expectedly, the absorption cross section is lower for both dyes when excited with longer wavelengths because there are fewer number of fluorophores with high-enough excitable vibrational states in the excited volume Fig. . The mea− 2 M and 10− 2 M methanol solutions, respectively. The signal from the sample was coupled into a multimode fibre using a reflective collimator at the camera port and measured with an OceanOptics QE65000 spectrometer.In the second experiment, the emission spectra of Stokes and anti-Stokes fluorescence were recorded. Continuous wave laser sources (405 and 634 nm) were used for excitation with two dichroic mirror and emission filter sets (Chroma ZT458rdc + Chroma ET460lp and Semrock Di01-R405/488/532/635 + Semrock FF01-446/-510/581/703). Rh101 and AF568 solutions were prepared in 10S1 to S0.The results show that the normalized shape of the Stokes and anti-Stokes emission spectra highly overlap Fig. . Only a − 5 M solution of methanol and PBS (1:1) to minimize the solvent’s evaporation between the slide and cover glass. To thermally isolate the sample from the microscope frame, a dry objective (Nikon CFI S Plan Fluor ELWD 20XC) was used throughout these experiments. For excitation, a 640 nm wavelength pulsed laser with a repetition rate of 20 MHz was used in a confocal line-scanning mode. The focal plane was set to approximately 1 μ m above the cover glass and was maintained by the Nikon perfect focus system. The temperature was changed gradually between 12 ∘C and 48 ∘C with a step size of 4 ∘C. After the sample reached thermal equilibrium , a 60 s measurement was recorded in the two emission channels (600/50 and 690/70). The measurements were repeated several times and averaged. These multiple passthroughs over the entire temperature range in both directions eliminated the possible hysteresis effects and photobleaching. The measurements with AF568 dye were slightly affected by photobleaching after multiple heating–cooling cycles. This effect was not observable for the Rh101 dye.A third experiment aimed to measure the temperature-dependence of the absorption cross section. A thermal paste was applied to a glass slide, and the slide was glued to a Peltier-cooled mount (Thorlabs TCLDM9). Each dye sample was prepared in a 10A strong increase in anti-Stokes emission as a function of temperature was observed Fig. . Accordi5. The arrival time distribution was fitted with a single exponential model using the Tail Fit method .Fluorescence lifetimes for Stokes and anti-Stokes emissions were measured with 560 nm and 640 nm wavelength excitations. To avoid nonlinear effects and detector saturation, neutral filters with OD3 and OD1 were used in the excitation path. The repetition rate of the pulsed laser sources was set to 20 MHz. The samples from the first experiment were reused, and four FLIM measurements were recorded in the 600/50 emission channel. The acquisition was stopped when the collected number of emitted photons exceeded 10The fitted lifetimes for the anti-Stokes fluorescence  were found to be consistent with those for the Stokes fluorescence   laser power in the far-red region can significantly increase the fluorescence background in the ”green channel”. This undesirable effect is typically mitigated with additional emission filters. However, the problem can be dealt with on the excitation side too, either by choosing an excitation laser with a higher wavelength . However, the density and spatial distribution of fiducial markers on the sample cannot be controlled. They also introduce a high intensity light source, precluding the localization of adjacent single dye emitters. Fluorescent beads labelled with hot-band active dyes (e.g. Rh101 or AF568) can be used to address this issue. The lateral drift can be corrected by simultaneously recording the anti-Stokes signal in a separate spectral channel. A similar approach using only one emission window has been already proposed . In addim2) and finite lifetime of the switching buffer (< 4 hours) in localization based microscopy methods make the selection of ROI a difficult task. Labelling the interesting cells, cell components, structures or proteins in the sample with a different dye can be used to significantly reduce searching time. Hot-band active dyes are an ideal choice for such applications since the two spectral channels can be captured simultaneously using the same laser source. For example, the axon initial segment of a neuron in a brain tissue experiment can be found by labelling the βIV spectrin [The reduced FOV (< 20 × 20 μspectrin . In a muspectrin , 30, whiAutofluorescence is a critical limitation in fluorescence microscopy. It reduces the signal-to-noise ratio and can introduce artefacts. Several sample preparation protocols and histochemical techniques  have beeComparative time-resolved and steady-state measurements of the anti-Stokes fluorescence emission of AF568 have revealed that the emission intensity linearly depends on both the excitation power and temperature in the studied ranges. No intensity threshold was detected and both the emission spectrum and the fluorescence lifetime were found to be identical to the normal Stokes fluorescence values. These findings have confirmed that the detected anti-Stokes fluorescence of AF568 is the consequence of the hot-band absorption-emission pathway. The undesirable anti-Stokes emission rate can be reduced by manipulating either the temperature or the excitation wavelength. However, several potential applications have been suggested where hot-band active dyes can be used for drift correction, fast ROI selection or autofluorescence reduction.


This study was designed to investigate the effects and underlying mechanisms of Astaxanthin (AST) on high-fructose-induced hyperuricemia (HUA) from the perspectives of the uric acid (UA) synthesis and excretion in rat models. Following six weeks of a 10% fructose diet, the level of serum UA effectively decreased in the AST groups as compared to the model group. The enzymatic activities of xanthine oxidase (XOD) and adenosine deaminase (ADA) were significantly inhibited, and the mRNA expression levels of XOD and ADA significantly decreased after the AST administration. These results suggested that the AST reduced UA synthesis by inhibiting the mRNA expressions and enzyme activities of XOD and ADA, thereby contributing to HUA improvement. On the hand, the relative expressions of the mRNA and protein of kidney reabsorption transport proteins (GLUT9 and URAT1) were significantly down-regulated by AST, while that of the kidney secretion proteins  were significantly up-regulated by AST. These results indicated that the AST promoted UA excretion by regulating the urate transport proteins, and thus alleviated HUA. This study suggested that the AST could serve as an effective alternative to traditional medicinal drugs for the prevention of fructose-induced HUA. Fructose, a naturally occurring monosaccharide present in fruit, honey, and some vegetables, has plenty of use in processed foods . In receThe accumulated UA is partially excreted out of body by blood circulation, while approximately 70% of the UA excretion occurs through the kidney . The excAster glehni inhibited XOD activity and its administration decreased the serum UA level in potassium oxonate-induced HUA rats [Alpinia oxyphylla seed extract improved the reabsorption and secretion of UA in kidney by regulating the UA transport proteins , which led the attenuation of HUA in rat models [Currently, the cornerstone in the prevention of HUA involves the exploitation of drugs, which can promote the excretion of UA or block its synthesis . AllopurHUA rats . Wang etHUA rats . Besidest models .Astaxanthin (Ast), a xanthophyll carotenoid, is mainly derived from the marine organisms , and hasThe high-fructose diet is a major incentive in the case of HUA . 10% fruBlocking the UA synthesis is a key strategy for the treatment of HUA, since the reduction in UA synthesis directly leads to the decrement of serum UA level . XOD andAs illustrated in The under-excretion of UA in kidney is another main cause of HUA . In ordeThe level of serum UA is known to be strongly associated with the excretory function of kidney . The excAfter the glomerular filtration, UA undergoes the processes of reabsorption and secretion in renal tubules, where almost 90% of the UA is reabsorbed into the blood, while the rest is excreted in urine . The excOAT1 and OAT3 are mainly involved in the secretion of UA into the renal proximal tubules, while their specific function is to secrete UA into the epithelial cells through the basolateral membrane . In the Astaxanthin was offered by Jingzhou Natural Astaxanthin Inc. . Allopurinol was purchased from Aladdin . Fructose (99%) was offered by Shanghai Macklin Biochemical Technology Co., Ltd. . The rabbit anti-rat polyclonal primary antibodies for URAT1, GLUT9, OAT1, OAT3, ABCG2, β-actin and horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody were provided by Beijing Bioss Biological Technology Co., Ltd. . The kits for UA, XOD, ADA, Cr and BUN were purchased from Jiancheng Bioengineering Institute . HiScript III RT SuperMix for qPCR (+gDNA wiper) was purchased from Nanjing vazyme Biological Technology Co., Ltd. . TRIzol reagent was purchased from Shanghai Thermo Fisher . All reagents used in this study were of analytical grade.The experiments on rats were conducted according to the Animal Research Committee of Zhejiang Ocean University, . The experiments were conducted on male Sprague-Dawley (SD) rats with body weight around 230 ± 10 g, purchased from Experiment Animal Center of Zhejiang Province . All the animals were housed in a controlled atmosphere (22 ± 2 °C at 55 ± 5% humidity) with normal light:dark (12:12 h) cycle and free access to food and water. The rats were supplied with water and regular diet and libitum for 7 days of acclimation before starting the study.n = 8/group), which included: (1) control group (Control); (2) model group (Model); (3) allopurinol group (ALL), in which rats were orally administered with allopurinol (40 mg/kg BW/day); (4) astaxanthin low dose group (ASL-L), in which rats were orally administered with astaxanthin (10 mg/kg BW/day); (5) astaxanthin middle dose group (ASL-M), in which rats were orally administered with astaxanthin (20 mg/kg BW/day) and (6) astaxanthin high dose group (ASL-H), in which rats were orally administered with astaxanthin (40 mg/kg BW/day). The control group received normal drinking water, while the other groups were given drinking water with 10% fructose for six weeks to induce HUA. On day 40, all the rats were singly housed into metabolic cages to collect 24 h urine. At the end of sixth week, the rats were sacrificed and the blood samples were collected for serum preparation. The kidney and liver were isolated and stored at −80 °C for the subsequent experimentations.Rats were divided into 6 groups  using SPSS version 23 software .In this study, the data was presented as mean ± SD (standard deviation). The statistical significance between groups was determined by one-way ANOVA, followed by the Tukey–Kramer test (The present study investigated the preventive effects of AST on high-fructose-induced hyperuricemia (HUA) in rats. After AST administration, the level of serum UA significantly decreased as compared to the model group. In particular, the high dose of AST restored the serum UA level to normal. From the aspect of UA synthesis, the AST decreased the enzymatic activities of XOD and ADA in serum and the liver by down-regulating the mRNA expression of XOD and ADA in the liver. Meanwhile, the AST also down-regulated the expressions of the mRNA and protein of reabsorption proteins (URAT1 and GLUT9), as well as up-regulated the expressions of the mRNA and protein of secretion proteins . Based on these results, it is indicated that the AST decreased the serum UA level by reducing the UA synthesis and promoting the UA excretion. Therefore, the AST could serve as a potential agent for the prevention of HUA.


Oncogenic activation of the phosphatidylinositol-3-kinase (PI3K), protein kinase B (PKB/AKT), and mammalian target of rapamycin (mTOR) pathway is a frequent event in prostate cancer that facilitates tumor formation, disease progression and therapeutic resistance. Recent discoveries indicate that the complex crosstalk between the PI3K-AKT-mTOR pathway and multiple interacting cell signaling cascades can further promote prostate cancer progression and influence the sensitivity of prostate cancer cells to PI3K-AKT-mTOR-targeted therapies being explored in the clinic, as well as standard treatment approaches such as androgen-deprivation therapy (ADT). However, the full extent of the PI3K-AKT-mTOR signaling network during prostate tumorigenesis, invasive progression and disease recurrence remains to be determined. In this review, we outline the emerging diversity of the genetic alterations that lead to activated PI3K-AKT-mTOR signaling in prostate cancer, and discuss new mechanistic insights into the interplay between the PI3K-AKT-mTOR pathway and several key interacting oncogenic signaling cascades that can cooperate to facilitate prostate cancer growth and drug-resistance, specifically the androgen receptor (AR), mitogen-activated protein kinase (MAPK), and WNT signaling cascades. Ultimately, deepening our understanding of the broader PI3K-AKT-mTOR signaling network is crucial to aid patient stratification for PI3K-AKT-mTOR pathway-directed therapies, and to discover new therapeutic approaches for prostate cancer that improve patient outcome. Prostate cancer is the second leading cause of cancer-related deaths in men worldwide, despite extensive efforts to raise awareness and significant advancements in detection, screening, and treatment approaches ,2,3. AltPIK3CA, PIK3CB and PIK3CD respectively) and a regulatory subunit  that controls protein localization, receptor binding, and activation . PT. PT24]. nic mice . SLC43A1) to maintain sufficient levels of leucine needed for mTORC1 signaling and cell growth  loss of function mutations that lead to the stabilization of the SPOP substrate SRC3 , which consequentially increases PI3K activity [SPOP is frequently mutated (9–11% incidence) [SPOP mutation can also stabilize AR and potentiate AR signaling whilst the PI3K-AKT-mTOR signaling pathway is activated, allowing coordinated and cooperative signaling that drives tumorigenic growth [The reciprocal feedback loop between AR and PI3K-AKT-mTOR signaling may also be perturbed by Speckle-type BTB/POZ protein  ,46. Remac growth . Conversc growth . Furtherc growth , furtherPTEN loss or AKT activating mutations, low/absent AR-V7 protein levels and elevated p-ERK [Taken together, these findings support the rationale for combining pharmacological inhibition of the AR and PI3K-AKT-mTOR cascades to treat prostate cancer in the clinic, and highlight the need for further work to delineate the molecular mechanisms underpinning crosstalk between these two oncogenic cascades. Importantly, clinical trials exploring co-targeting AR and PI3K-AKT-mTOR signaling are beginning to show promise. A randomized Phase Ib/II study combining the pan-AKT inhibitor ipatasertib with abiraterone in mCRPC patients has reported ipatasertib + abiraterone prolongs radiographic progression-free survival (rPFS), improves overall survival and extends time to PSA progression compared to abiraterone alone, particularly in patients with PTEN loss  . This sted p-ERK . Neverthed p-ERK ,315,316.ed p-ERK , presentMYC and AXIN2 [2+ that stimulates calmodulin-dependent kinase II (CamKII), calcineurin and protein kinase C (PKC) signaling (WNT/Ca2+ pathway) [The WNT family is an evolutionarily conserved group of proteins essential for growth control, organ development, tissue homeostasis and stem cell renewal in multiple organs, and is crucial for normal prostate development ,319. WNTnd AXIN2   .APC deep deletion/truncating mutations and CTNNB1/β-catenin activating mutations [AR is in fact a WNT/β-catenin target gene, and AR and β-catenin can directly interact and co-localize in the nucleus to mediate transcriptional activity of AR-regulated genes [Activation of both the canonical and non-canonical WNT cascades has been reported in localized and advanced prostate cancer, and oncogenic deregulation of core WNT pathway components frequently occurs in primary and metastatic prostate cancer (up to 6% and 19% incidence respectively ), primarutations ,322,323.utations ,326,327,ed genes ,329,330 ed genes .Apc bi-allelic deletion predisposes to prostate adenocarcinoma in mice [Pten heterozygous or homozygous deletion to promote prostate cancer progression, CRPC transition and metastatic potential [Mouse models have been instrumental in determining the role of WNT signaling in prostate cancer, and we and others have shown that constitutive activation of β-catenin or  in mice ,332,333.otential ,332,333,Several molecular mechanisms that permit cross-regulation of the PI3K-AKT-mTOR and WNT signaling cascades have emerged in the literature, which could influence prostate cancer growth and resistance to anti-androgens and/or PI3K-AKT-mTOR pathway inhibitors ,334,335 In addition to transmitting canonical WNT signals, β-catenin also form adherens junctions with α-catenin and E-cadherin at cell–cell junctions to maintain tissue architecture and facilitate cell–cell signaling . PTEN haCrosstalk between the PI3K-AKT-mTOR and WNT/β-catenin signaling pathways is also mediated via GSK3β and the TSC1:TSC2 complex  Figure . GSK3β pPI3K-AKT-mTOR and WNT signaling may also interact through Hippo signaling. Hippo signaling is tightly intertwined with cell size regulation and nutrient sensing through LKB1-AMPK, TSC1:TSC2 and mTOR ,347, andNon-canonical WNT signaling has also been shown to activate the PI3K-AKT-mTOR pathway. For example, WNT/FZD7-dependent dissociation of Gβγ from Gαi enhances PI3K-AKT signaling and increases tumor cell invasive potential . ROR1 caMYC is a WNT/β-catenin target gene [MYC mRNA stability, translation and protein stability [MYC to maintain normal homeostasis [Interestingly, the PI3K-AKT-mTOR, WNT, MAPK and AR signaling cascades all converge to regulate the transcription factor MYC, which is frequently amplified in prostate cancer/mCRPC ,45,354. get gene , PI3K-AKtability ,359,360.eostasis ,362. Howeostasis . MYC upreostasis .AXIN2 [WNT inhibitors are beginning to enter clinical trials, including small-molecule inhibitors to the enzyme porcupine that block WNT ligand secretion, such as WNT974 (LGK974) ,322. PreAXIN2 , and recAXIN2 ,365. β-cAXIN2 . Thus, fPTEN deletion and PIK3CA activating mutation), however our molecular understanding of how these events differentially mediate cell signaling programs is limited, and several genetic alterations remain to be studied functionally. Future work to gain novel insight into the functional consequence of these genetic alterations in prostate cancer is necessary to (a) identify passenger vs. driver alterations, (b) establish the ability of individual aberrations to synergize with additional oncogenic events, and (c) discover their mode of action during tumor growth, metastasis and therapeutic resistance. In conjunction with genomic and transcriptomic data, establishing the frequency and impact of post-transcriptional modifications and epigenetic events within core PI3K-AKT-mTOR pathway components during prostate tumorigenesis and disease progression/recurrence is also crucial, as these components are regulated at multiple levels and genomic/transcriptomic data do not consistently equate with protein activity.In summary, the PI3K-AKT-mTOR cascade is frequently activated in prostate cancer, and genomic profiling has revealed that oncogenic genetic alterations occur within a diverse array of PI3K-AKT-mTOR pathway components. Significant research efforts have been devoted to delineating the mode of action of several of these aberrations (e.g., Targeting the PI3K-AKT-mTOR pathway in prostate cancer remains a key clinical challenge. Therapeutic resistance emerges owing to various feedback/feedforward loops and redundancy mechanisms that prevent complete suppression of the pathway and cause compensatory augmentation of interacting signaling pathways, thus rationalizing the exploration of combination therapies. Encouragingly, clinical trials are beginning to report therapeutic efficacy when combining PI3K-AKT-mTOR and androgen/AR-directed therapies, particularly in patients with mCRPC that display PTEN loss. However, the mechanism of resistance to PI3K-AKT-mTOR pathway-targeted therapies is likely to vary dramatically between patients and within individual tumors owing to several factors. These include the activity status of the pathway components, the extent of intratumoral heterogeneity, the mode and concentration of upstream stimuli, the genetic alterations present and the composition of the tumor microenvironment. Furthermore, our ability to successfully translate preclinical findings to the clinic is currently hampered by the limited number of prostate cancer preclinical models available, which do not fully cover the broad range of prostate cancer subtypes or disease heterogeneity seen in the clinic. Accordingly, to discover newtherapeutic approaches that increase patient response rates and overall survival, further delineation of the complex signaling network that exists within the PI3K-AKT-mTOR pathway and the interacting MAPK, AR, and WNT pathways is needed, together with the development of a wider range of preclinical models that better recapitulate the clinic and a deeper understanding of the molecular biology underpinning prostate cancer disease subtypes and tissue heterogeneity.