Further analysis demonstrated that FAK activates PI3K through the association and tyrosine phosphorylation of the p85 regulatory subunit of PI3K and that UM cells require PI3K/AKT signaling for survival. These findings establish a novel link between Gαq-driven signaling and the stimulation of PI3K as well as demonstrate aberrant activation of signaling networks underlying the growth and survival of UM and other Gαq-driven malignancies.G proteins and G protein–coupled receptors activate a diverse array of signal transduction pathways that promote cell growth and survival. Indeed, hot spot–activating mutations in GNAQ/GNA11, encoding Gαq proteins, are known to be driver oncogenes in uveal melanoma (UM), for which there are limited effective therapies currently available. Focal adhesion kinase (FAK) has been recently shown to be a central mediator of Gαq-driven signaling in UM, and as a result, is being explored clinically as a therapeutic target for UM, both alone and in combination therapies. Despite this, the repertoire of Gαq/FAK-regulated signaling mechanisms have not been fully elucidated. Here, we used a whole-genome CRISPR screen in GNAQ-mutant UM cells to identify mechanisms that, when overactivated, lead to reduced sensitivity to FAK inhibition. In this way, we found that the PI3K/AKT signaling pathway represented a major resistance driver. Our dissection of the underlying mechanisms revealed that Gαq promotes PI3K/AKT activation  GNAQ and GNA11, referred to as GNAQ oncogenes, encoding GTPase-deficient and constitutively active Gαq proteins, have been identified in ∼93% of uveal melanoma (UM) and 4% of skin cutaneous melanoma, where they act as driver oncogenes (G protein–coupled receptors (GPCRs) and their associated G proteins are the largest family of cell surface proteins involved in signal transduction. As a result, they are central mediators of numerous cellular and physiological processes , 2. Mostncogenes , 9, 10.UM is the most common primary cancer of the eye in adults and is the second most common melanoma subtype after skin cutaneous melanoma . ApproxiGNAQ oncogene . Toward this end, we aimed at investigating additional Gαq-FAK-regulated signaling circuitries that may be critical to promote growth in UM and other Gαq-driven malignancies.GNAQ-mutant UM cells in the context of FAK inhibition (A). Using Cas9-expressing 92.1 UM cells (92.1Cas9), infected with the Brunello Human Genome pooled single-guide RNA (sgRNA) library, cells were passaged under 0.1 μM VS-4718 (FAKi) treatment, or vehicle for a total of 19 cell doublings. In order to evaluate pathways that when modulated, resulted in resistance to inhibition of FAK, we examined sgRNAs enriched in FAKi treatment condition. Among the top hits were phosphatase and tensin homolog (PTEN) and tuberous sclerosis complex 2 (TSC2), which are canonical negative regulators of the PI3K/AKT pathway (B). Aligned with this, genes involved in the PI3K/AKT/mammalian target of rapamycin (mTOR) signaling pathway were enriched targets of the sgRNAs yielding the most resistance . We also observed enrichment of cells with AMOTL2 sgRNAs in the FAKi conditions, which is a negative regulator of the Hippo/YAP pathway, and is aligned with the role of YAP as a downstream target of FAK in UM (p = 0.03) (D). To validate the findings of our CRISPR screen, we performed siRNA-mediated knockdown of the top two PI3K/AKT pathway hits from our screen, PTEN and TSC2, and evaluated the effect on cell viability in response to FAK inhibition (E). We found that knockdown of PTEN and TSC2 both resulted in decreased sensitivity to FAKi in UM cells. We next evaluated the effect on PI3K and FAK signaling caused by PTEN and TSC2 loss . In both cases, while siRNA-mediated knockdown of PTEN and TSC2 resulted in increase in phosphorylation of downstream pathway members, AKT and S6 respectively, the latter often used to monitor mTOR activity  D. To valhibition E. We fouSC2 loss F, S1B. Iactivity , it did A and B, A–D). However, we did not observe a decrease in the same signaling targets upon pharmacological inhibition of Gαq in a non-Gαq-dependent cutaneous melanoma cell line, SKMEL-28 (E). This suggests that Gαq controls PI3K signaling in UM cells harboring active Gαq. As an orthogonal approach, we found that GαqQL, the active Gαq mutant found in UM, promoted the accumulation of the phosphorylated forms of ERK (pERK), FAK (pFAK), AKT, and S6 in human embryonic kidney 293 (HEK293) cells, demonstrating the direct ability of Gαq to promote PI3K/AKT signaling . We also challenged our observations using the expression of a synthetic Gαq-coupled receptor, termed Gαq-DREADD, which can only be activated by addition of a pharmacologically inert ligand, clozapine-N-oxide (CNO) (D). We validated the specificity of this approach by expressing Gαq-DREADD in Gαq/11 KO cells and stimulating with CNO; however, we did not observe an increase in the phosphorylation state of any of the proteins tested (E). Challenging both HEK293 and HEK293 Gαq/11 KO cell lines with epidermal growth factor (EGF) treatment revealed an increase in phosphorylation of all tested proteins in both cases, demonstrating the signaling competence in both models (F). Collectively, these results indicated that Gαq promotes PI3K/AKT signaling when activated by GPCRs or as part of constitutive Gαq signaling, such as in UM.In this case, however, whether Gαq activates or inhibits PI3K/AKT is not clear, and the overall underlying mechanisms involved are poorly understood , 27, 28.SKMEL-28 E. This signaling C, S2F. Wde (CNO) . Expressde (CNO) D. We vals tested E. Challeh models F. Collecvia FAK. To test this, we expressed GαqQL in HEK293 cells alone or in combination with pharmacological inhibition of FAK (A). Indeed, inhibition of FAK in the context of Gαq activation was sufficient to block an increase in pAKT and pS6, whereas no change in pERK was observed. Likewise, activation of Gαq using Gαq-DREADD and stimulation with CNO, in combination with FAK inhibition, abrogated an increase in pAKT and pS6 (B). Based on these findings, we tested the ability of FAK expression to drive PI3K/AKT signaling. Overexpression of FAK in HEK293 cells led to a potent increase in pAKT and pS6 . Conversely, blockade of FAK in UM cells with high basal Gαq-FAK activity, using siRNA-mediated knockdown, or by a pharmacological inhibition led to a decrease in pAKT and pS6 levels . These data suggest that in UM cells, persistent Gαq-driven signaling promotes PI3K pathway signaling via FAK.Based on these findings linking Gαq to enhanced PI3K/AKT activity, we then asked whether Gαq controls PI3K/AKT signaling n of FAK A. Indeed and pS6 B. Based  and pS6 C, S3A. Cels D–F, , B–D. ThG). The reverse could also be observed, where under basal conditions, co-IP of p85 revealed strong association with FAK that was relieved upon FAKi treatment . We also observed strong basal tyrosine phosphorylation of p85 that was diminished concomitant with a dissociation from FAK by FAKi treatment. We validated our findings by global IP of tyrosine-phosphorylated proteins using pTyr antibodies in UM cells . Aligned with our previous results, IP of total pTyr resulted in pulldown of p85 and FAK. Inhibition of FAK activity with FAKi similarly reduced the levels of tyrosine pFAK and p85 available to be extracted by pTyr. Taken together, these findings suggest that Gαq signaling promotes PI3K/AKT pathway activity through FAK-dependent tyrosine phosphorylation and association with PI3K-p85 .The p110 catalytic subunit of the PI3K heterodimer is comprised of four different isoforms: PI3Kα, PI3Kβ, PI3Kγ, and PI3Kδ. Class IA PI3Ks  consist of heterodimers of a catalytic p110 subunit and regulatory p85 subunit . In respFig. 3A). We next performed siRNA-mediated knockdown of the major UM-associated p110 isoforms alone, and in combination, and assessed levels of PI3K pathway activity by measuring pAKT and pS6 (B). We found that in UM cells, PI3Kα and PI3Kβ were major drivers of PI3K signaling, with the strongest reduction in the context of triple p110 isoform knockdown. To complement our genetic knockdown approach, we tested the ability of p110 isoform-specific as well as a pan-PI3K pharmacological inhibitor to inhibit AKT/S6. Aligned with our knockdown data, only BKM120, the pan-PI3K inhibitor that we tested, was able to reduce both pAKT and pS6 in a potent and sustained manner, in comparison to inhibitors targeting individual p110 isoforms (C) (D and E) and induction of apoptosis , indicating that UM cells are reliant on PI3K signaling for growth and survival. Ultimately, these results expand the repertoire of Gαq-FAK-regulated signaling circuitries and establish a direct connection between Gαq and PI3K/AKT via FAK (G).While expression patterns of each PI3K catalytic subunit isoforms varies across tissues, the expression and isoform usage of PI3K is not currently known in UM. We first screened expression of each PI3K-p110 isoform in a number of UM cell lines on the DepMap portal and found that with the exception of PI3Kγ, all p110 isoforms were expressed A. We nex and pS6 B. We fouisoforms C 31). F. FA). Weorms (C) , D and Epoptosis F, S4A, i via FAK G.Figure GNAQ oncogene is the major oncogenic driver for UM, a cancer type characterized by limited additional genetic aberrancies. As a result, UM serves as a unique model to interrogate and profile the diversity of signaling mechanisms initiated by Gαq and Gαq-coupled GPCRs to promote cell proliferation. Coupled with this, a deeper understanding of Gαq-initiated mitogenic networks provide an opportunity for the identification of novel signal transduction–based targeted therapies against UM.The Our dissection of the signaling networks regulated by Gαq led to the finding that activation of Gαq is sufficient to promote PI3K pathway. Further interrogation into the underlying mechanisms revealed that Gαq controls PI3K activation through FAK-mediated association and phosphorylation of the p85 regulatory subunit of PI3K. Finally, we demonstrate that UM cells are sensitive to genetic and pharmacological inhibition of PI3K signaling. Taken together, these findings revealed a novel signaling axis by which Gαq controls cell growth and survival by regulating the PI3K/AKT/mTOR pathway through FAK.in vitro and preclinical models of UM, but the molecular basis for these findings, as well as whether GNAQ activates the mTOR pathway, has not been fully investigated  containing 10% fetal bovine serum , 1× antibiotic/antimycotic solution , and 1× Plasmocin prophylactic . HEK293 Gαq/11 KO cells were cultured using the same conditions described previously and were a kind gift from Dr Asuka Inoue . UM cellPlasmids pCEFL-HA, pCEFL-HA-GαqQL, and pCEFL-HA-Gαq-DREADD were described previously . pEGFP-CCas9). The human Brunello whole-genome CRISPR pooled library was a gift from David Root and John Doench (Addgene #73178). The library contains 76,441 sgRNAs targeting 19,114 genes (four sgRNAs per gene) and 1000 nontargeting sgRNAs as the negative control.Genome-wide CRISPR-KO screen was performed using the methods described . BrieflyCas9 cells into 2245 mm × 245 mm tissue culture dishes plates (12 × 106 cells/plate) divided into two treatment arms: three replicate plates for either vehicle/dimethyl sulfoxide or VS-4718 treatments. A total of 24 × 106 cells were passaged into new plates containing dimethyl sulfoxide or 0.1 μM VS-4718 until the population doubling level reached 19. A total of 24 × 106 cells were aliquoted from each plate at the end of the screen and stored at 80 °C for genomic DNA extraction and subsequent sgRNA quantification. The entirety of isolated genomic DNA was used for subsequent PCR to ensure capturing the full representation of the libraries. PCR products were sequenced on a HiSeq4000 instrument (Illumina) (350 million reads).The screen was performed by seeding 92.1versus control samples. Over-representation analysis of top resistance driving hits against Kyoto Encyclopedia of Genes and Genomes  . Read co Genomes  and Bioc Genomes  pathways Genomes , 49. P V50) were determined from the curve using the nonlinear log (inhibitor) versus response–variable slope (three parameters) equation. GI50 values were only determined for compounds that inhibited growth by more than 50%.Cells were seeded at a density of 8000 cells/well in 96-well white plates. Eight dilutions of each inhibitor were assayed in technical triplicates for 72 h. Cell viability was measured with the AquaBluer Cell Viability Reagent on a Spark microplate reader (Tecan). Using the GraphPad Prism, version 8.2.0 software , the half-maximal inhibitor concentration values  supplemented with Halt Protease and Phosphatase Inhibitor Cocktail  and 1 mM sodium orthovanadate . Lysates were centrifuged at maximum speed at 4 °C, concentrations were measured using DC Protein Assay , and lysates were prepared with addition of 4× Laemmli Sample Buffer  and boiled for 5 min at 98 °C.3VO4, protease/phosphatase inhibitor [Thermo Scientific], 1 mM DTT, and 1 mM PMSF) and centrifuged at 16,000g for 5 min at 4 °C. Supernatants were incubated with primary antibody overnight at 4 °C, and protein A conjugated Sepharose beads for 1 h at 4 °C. Beads were washed three times with lysis buffer and prepared with addition of 4× Laemmli Sample Buffer  and boiled for 5 min at 98 °C.For IPs, cells were lysed with IP lysis buffer  bovine serum albumin or 5% w/v skim milk. Primary antibodies were all purchased from Cell Signaling Technologies and used at 1:1000 dilution. FAK , pY397-FAK , PTEN , TSC2 , AKT , pS473-AKT , S6 , pS235/236 S6 , ERK1/2 , pT202/Y204-ERK1/2 , GAPDH , beta-actin , vinculin , p-Tyrosine , p85 , p110α , p110β , and p110δ . Horseradish peroxidase–conjugated goat anti-rabbit and antimouse immunoglobulin G (Southern Biotech) were used at a dilution of 1:30,000, and immunoreactive bands were detected using Immobilon Western Chemiluminescent horseradish peroxidase substrate (Millipore) according to the manufacturer’s instructions.Cells were seeded at a density of 10,000 cells/well in 96-well white plates. After 24 h, drug treatment or vehicle was added, and cells were assayed as indicated. Apoptosis was measured using the Promega CaspaseGlo3/7 Assay System (G8090) as per the manufacturer’s instructions.t test . All experiments were repeated independently with similar results at least three times.All data analyses were performed using GraphPad Prism, version 9.4.0 for Mac. The data were analyzed by one-way ANOVA test with correction for multiple comparison or sgutkind@health.ucsd.edu).All data associated with this study are presented within the article. CRISPR screen sequencing files have been deposited to the National Center for Biotechnology Information Sequence Read Archive under the BioProject accession number: PRJNA902794. Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr J. Silvio Gutkind (This article contains O.H. is an employee of Zentalis Pharmaceuticals, D.N. is an employee of TwinStrand Biosciences, unrelated to this study. P.M. is a scientific cofounder of Shape Therapeutics, Boundless Biosciences, Navega Therapeutics, and Engine Biosciences. The terms of these arrangements have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies. J.S.G. is consultant for Domain Therapeutics, Pangea Therapeutics, and io9, and founder of Kadima Pharmaceuticals, outside the submitted work. All other authors declare that they have no conflicts of interest with the contents of this article.


Human T cell leukemia virus type 1 (HTLV-1), the etiological agent of adult T cell leukemia/lymphoma (ATLL) and of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), was identified a few years before Human Immunodeficiency Virus (HIV). However, forty years later, our comprehension of HTLV-1 immune detection and the host immune responses to HTLV-1 is far more limited than for HIV. In addition to innate and adaptive immune responses that rely on specialized cells of the immune system, host cells may also express a range of antiviral factors that inhibit viral replication at different stages of the cycle, in a cell-autonomous manner. Multiple antiviral factors allowing such an intrinsic immunity have been primarily and extensively described in the context HIV infection. Here, we provide an overview of whether known HIV restriction factors might act on HTLV-1 replication. Interestingly, many of them do not exert any antiviral activity against HTLV-1, and we discuss viral replication cycle specificities that could account for these differences. Finally, we highlight future research directions that could help to identify antiviral factors specific to HTLV-1. Human T cell leukemia virus type 1 (HTLV-1) is a Deltaretrovirus estimated to chronically infect 5–10 million people worldwide, a number that is probably underestimated ,3. In adUpon chronic infection, the major cellular target of HTLV-1 is CD4+ T cells, in which the viral oncogenic process can result in the development of leukemia/lymphoma. Other immune cells can also be infected, such as CD8+ T cells and myeloid cells. In particular, dendritic cells (DC) have been suggested to act as the first cells encountered during primo-infection . DC can As a retrovirus, the HTLV-1 genome is integrated in the host cell genome. HTLV-1 chronic infection has been shown to be maintained by clonal proliferation of infected CD4+ T cells  9]. Thi. Thi9]. In addition to innate and adaptive immune responses that rely on specialized cells of the immune system, host cells may also express a range of antiviral factors that inhibit viral replication at different stages of the cycle, in a cell-autonomous manner. These antiviral factors can be constitutively expressed, and therefore act as immediate intrinsic barriers to viral replication. However, most of the antiviral factors are inducible upon the innate sensing of viral infection by pattern recognition receptors, as they are encoded by type-I interferon (IFN-I)-stimulated genes (ISG). This feature of antiviral factors thus bridges intrinsic and innate immunity, as IFN-I can be produced in large amounts by specialized innate cells such as plasmacytoid dendritic cells (pDC) and myeloid cells.Cell-autonomous antiviral factors were originally discovered in the HIV field, and multiple HIV-directed antiviral factors have been identified since then, belonging to diverse protein families and exerting antiviral activities through diverse mechanisms ,17. A suBeing close to the lentivirus HIV in terms of viral organization, tropism and viral replication cycle, it is tempting to speculate that HTLV-1, although belonging to a different retroviral genus, might also be controlled by restriction factors with demonstrated antiviral activity against HIV. However, while the infection of CD4+ T cells by HIV leads to the massive release of neo-synthesized viral particles, and to the death of the infected cell, infection by HTLV-1 leads to the rapid shut-down of viral particle production, and induces CD4+ T cell spontaneous proliferation that can progress to cell immortalization and transformation. Hence, while host T cells might be under a strong selection pressure to restrict HIV replication cycle, this might not be applicable to host T cells exposed to HTLV-1. In this work, we review the extent to which HIV restriction factors can restrict HTLV-1 replication . As expeAs stated in the introduction, most HIV restriction factors are encoded by ISG ,21, explMost studies focused on the possible activity of IFN-I in the late stages of the viral replication cycle and in the proliferation of chronically infected T cells. In several in vitro studies, Kinpara et al. demonstrated that an IFN-α treatment suppressed HTLV-1 gene expression and the proliferation of HTLV-1 infected T cells ,23. SimiWe further showed that IFN-I-induced restriction of the late stages of HTLV-1 infection in T cells is mediated by Protein Kinase R (PKR), whose activation reduces the translation of HTLV-1 mRNA . PKR alsImportantly, IFN-I might differentially affect HTLV-1 infection depending on the cell type. Accordingly, the HTLV-1 auxiliary p30 protein is required for the infection of monocytes and dendritic cells, but not of T cells ,32, and Until now, no unbiased systematic screen for HTLV-1 restriction factors has been performed. Instead, most studies have analyzed the ability of known HIV restriction factors to restrict HTLV-1 replication. Because HTLV-1 shares a similar replication cycle with HIV, it is indeed tempting to hypothesize that HIV restriction factors are also active against HTLV-1. However, one should bear in mind that our understanding of some of the steps of the HTLV-1 replication cycle has been extrapolated from the HIV field, with no confirmation of the specific context of HTLV-1 infection. Therefore, the actual extent of similarity between the cycles of both viruses, in their molecular details, remains hypothetical. This holds true in particular for the early steps of uncoating, reverse transcription, nuclear transport and integration . In contINK4A and p27Kip1, and by activating positive regulators such as CDK4/6 and E2F  . Seminaliew, see ). HBZ waiew, see ,38. Tax iew, see ,40 Imporiew, see . This ociew, see ). NonethImportantly, several auxiliary proteins from HIV have demonstrated roles as antagonists of restriction factors, such as Nef or Vif, but these functions have not been identified in HTLV-1 proteins yet. This highlights the fact that the selection pressure exerted by host factors on both viruses might be very different, as will be discussed later in this review. In this section, we review the action or lack of action on HTLV-1 of some of the known HIV restriction factors, with a focus on TRIM family members, APOBEC3, SAMHD1, CIITA, ZAP and Tetherin . We furtThe tripartite motif (TRIM) family contains many interferon-induced proteins involved in restriction of several viruses, including lentiviruses . Among tNo direct interaction of TRIM5α with HTLV-1 capsid has been reported thus far, but one study performed on PBMC from HAM/TSP patients reported an association of TRIM5α polymorphisms with high proviral loads, suggesting that TRIM5α could also control HTLV-1 replication . This reFollowing these findings, other TRIM family members were investigated, and among them, TRIM19, also known as PML, was demonstrated to restrict HTLV-1 . This reAPOBEC3 proteins (A3) are a family of DNA cytidine deaminases (A3A to A3H), some of which display potent antiviral activity against HIV. Among them, A3G was the first restriction factor discovered for HIV. It is incorporated into the newly produced viral particles, and thus delivered to the target cell together with the viral genome. A3 act during reverse transcription where they induce G-to-A mutations in the viral DNA genome, thus affecting its integrity. Of note, A3 restriction may also occur without deaminase activity , by direInterestingly, although it is incorporated into HTLV-1 particles, A3G has no noticeable effect on HTLV-1 infectivity. Indeed, in vitro assays using HTLV-1 virion produced in the presence of ectopic A3G were capable of infecting T cell lines as the A3G-free virions and no G-to-A mutations were observed in the genome of cells infected with A3G-containing virions ,51,52,73SAMHD1 is another HIV restriction factor that targets pre-integration steps of the viral replication cycle in cells from the myeloid lineage. SAMHD1 is antagonized by the HIV-2-encoded protein Vpx ,74, whicThe effect of SAMHD1 on HTLV-1 is less clear. In DC and macrophages cultured in vitro, the degradation of SAMHD1 by SIV-Vpx delivered in target cells by lentivectors prior to infection did not affect HTLV-1 infection, while it efficiently increased that of HIV, suggesting that SAMHD1 is not active against HTLV-1 in DC and macrophages . In contHowever, in vivo monocytes from HAM/TSP patients or asymptomatic carriers are positive for integrated viral DNA, despite high expression of SAMHD1 . This coInterestingly, the simian foamy virus (FV), a retrovirus from the Spumavirus genus, is also resistant to A3G  and SAMHMHC Class II Transactivator CIITA presents a particular interest in the field of anti-viral immunity as it may affect viral infection through two main activities. First, CIITA positively controls the expression of Major Histocompatibility Class II (MHC-II) on antigen presenting cells , controlThe CIITA restriction of Deltaretroviruses was first investigated on HTLV-2, for which CIITA was demonstrated to exert antiviral activity in B and T cells . SimilarThese findings were observed following the ectopic expression of CIITA in 293T cells, but also in a promonocytic cell line, in which expression of CIITA is constitutive, strengthening its role in a more physiologic model. However, more research is needed to investigate whether CIITA is efficient in other myeloid cells, specifically in primary dendritic cells or monocytes that are most relevant to HTLV-1 infection.HIV, but also other retroviruses such as murine leukemia virus (MLV) or avian leukosis virus (ALV) ,91,92, aInterestingly, HTLV-1 transcripts were found to be particularly enriched in CpG sequences susceptible to ZAP-mediated suppression . In addiTetherin, also known as BST-2 , is a restriction factor active in the late stages of HIV infection. Tetherin inhibits viral particle release from the infected cell by retaining the particles at the cell surface ,94. OnceImportantly, the inability of Tetherin to restrict HTLV-1 could be well-explained by differences in the modes of dissemination of HTLV-1 versus HIV. While HIV efficiently uses both cell-free and cell-to-cell transmission, cell-free transmission does not significantly contribute to HTLV-1 dissemination, as stated above . BecauseIn vitro, HTLV-1 cell-to-cell transmission is highly efficient when the virus is present at the cell surface of the donor cell and concentrated in the form of the viral biofilm. Interestingly, Tetherin has been identified as a component of HTLV-1 viral biofilm , leadingOverall, these observations indicate that the antiviral activity of HIV restriction factors cannot be systematically extrapolated to HTLV-1, as some of them (such as A3G and Tetherin) fail to exert any antiviral activity against HTLV-1. In the absence of any known HTLV-1-encoded antagonists of these restriction factors, this lack of activity against HTLV-1 indicates that these restriction factors are not relevant in the HTLV-1 context. While the lack of activity of some restriction factors can be clearly explained by differences in the viral replication cycle of both viruses, as is the case of Tetherin and cell-free versus cell-to-cell viral transfer, the molecular basis for the lack of activity of other factors is still poorly understood. A better characterization of the HTLV-1 replication cycle, and in particular of the early steps that have not been specifically dissected, such as uncoating, reverse transcription, nuclear import and integration, might help to better understand the contrasting activity of restriction factors on both viruses.As stated above, antiviral restriction is intimately linked to innate immunity, as most restriction factors are induced by IFN-I upon viral sensing . Severalversus mature DC are known [Immature DC are permissive to HTLV-1 replication, although to a limited extend ,7. Howevre known , this ofPteropus alecto cells [As reviewed in the previous sections, only a handful of HIV restriction factors have been tested against HTLV-1. In fact, many more HIV restriction factors are known, and the list has continued to expand in the last decade, as exemplified by the discovery of the anti-HIV properties of IFITM, MxB or SERINC3/5 ,108,109,to cells . This ocBecause HIV is the most studied of human retroviruses, much HTLV-1 research has been inspired by discoveries made in the HIV field. However, widening the scope to other retroviruses aside from HIV might also prove useful  levels of infectious spread in vivo that may be compensated by clonal expansion of infected cells. This could explain the distinct pressure exerted by restriction factors on HIV and HTLV-1. Interestingly, the clearest data on restriction factors active against HTLV-1 relate to factors targeting viral expression, such as CIITA and ZAP. In fact, these factors could be interpreted as beneficial for HTLV-1, as they enforce a low level of viral expression, which is most adapted to the mode of persistence of HTLV-1 . This woInstead, searching for host factors that could inhibit HTLV-1 pro-proliferative action could represent an opportunity to identify HTLV-1-specific antiviral factors that could impede HTLV-1 clonal replication. Intriguingly, while HTLV-1 infects both CD4+ and CD8+ T cells and induces the clonal expansion of both cell types ,123, thePrimary CD4+ T cells isolated from infected individuals also show a heterogenous ability to undergo spontaneous proliferation ex vivo, which correlates with viral gene expression . In addiIn conclusion, while the classical concept of antiviral restriction against HTLV-1 might be rather inoperative in the chronic phase of infection, which shows low levels of infectious spread, the question of HTLV-1 restriction upon acute infection, which largely depends on infectious spread, remains open. Because DC are thought of as the first cells to become infected upon primo-infection, and because the infection of myeloid cells plays a crucial role in establishing HTLV-1 chronic infection ,31,132,


The advancement in high-throughput sequencing analysis and the evaluation of chromatin state maps have revealed that eukaryotic cells produce many non-coding transcripts/RNAs. Further, a strong association was observed between some non-coding RNAs and cancer development. The mitogen-activated protein kinases (MAPK) belong to the serine–threonine kinase family and are the primary signaling pathways involved in cell proliferation from the cell surface to the nucleus. They play an important role in various human diseases. A few non-coding RNAs associated with the MAPK signaling pathway play a significant role in the development of several malignancies, including liver cancer. In this review, we summarize the molecular mechanisms and interactions of microRNA, lncRNA, and other non-coding RNAs in the development of liver cancer that are associated with the MAPK signaling pathway. Further, we briefly discuss the therapeutic strategies for liver cancer related to ncRNA and the MAPK signaling pathway. Hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC), and combination hepatocellular carcinoma–cholangiocarcinoma (cHCC–CCA) are the three main pathophysiological subtypes of primary liver cancer. HCC accounts for 80–90%, and ICC accounts for 10–15% of all primary liver cancer cases . HCC is Mitogen-activated protein kinases (MAPKs) belong to the serine–threonine kinase family. The MAPK signaling pathway is primarily involved in cell proliferation from the cell’s surface to the nucleus . In eukaHOX transcript antisense RNA (HOTAIR), lncRNA highly upregulated in liver cancer (HULC), and telomerase RNA component (TERC). HOTAIR regulates the epigenetic silencing of HOXD sites [MIR-34 simulant used to treat patients with metastatic or advanced liver cancer by intravenous injection [The advent of high-throughput sequencing techniques and the elucidation of the chromatin state maps have helped identify several non-coding transcripts/RNAs produced by eukaryotic cells ,19,20. BXD sites , HULC acXD sites . In addinjection . TogetheMounting evidence has demonstrated a close correlation between the MAPK signaling pathway, ncRNA, and liver cancer. Further, ncRNA plays a significant role in the occurrence and progression of liver cancer. In this review, we summarize the molecular mechanisms of miRNA, lncRNA, and other ncRNAs associated with the MAPK signaling pathways in the progression of liver cancer, as well as their similarities and roles. We hope this review can shed light on the complex molecular network of the occurrence and development of HCC, and it could serve as the basis for developing therapeutic targets for liver cancer patients.To silence gene expression, the miRNAs, small ncRNAs with 22 nucleotides, drive the Argonaute (AGO) protein to the target of the 3’ untranslated regions (UTR) of mRNA . MAPK siOne of the most prominent kinase cascades in tumor cell biology is the Ras–Raf–MEK–ERK signaling pathway  . In mammSome miRNAs alter the expression of the ERK signaling pathway and vice versa, thus affecting the occurrence and progression of liver cancer. Certain microRNAs regulate the expression of Ras, which is involved in the MAPK signaling pathway.A study by Yang et al., showed a significant decrease in miR-140-5p levels in all six liver cancer cell lines and HCC tissues . FurtherApart from Ras, some microRNAs regulate the progression of liver cancers by targeting other proteins that regulate ERK1/2 phosphorylation.MAP4K4, which phosphorylates ERK1/2 by acting as a mediator for MEKK1 phosphorylation, thereby inhibiting NFAT5 expression. The decrease in NFAT5 expression inhibits NFAT5 binding to the aspartyl-tRNA synthetase 2 (DARS2) promoter, thereby promoting DARS2 (HCC oncogene) expression. This enhances cell cycle progression and inhibits the apoptosis of HCC cells. In vitro and in vivo studies have revealed that miR-320a acts as a tumor suppressor. MiR-320a interacts with PBX homeobox 3(PBX3) and increases the expression of matrix metallopeptidase 2 (MMP2) and cyclin-dependent kinase 2 (CDK2) by hindering the phosphorylation of ERK1/2, thus increasing the proliferation and metastasis of HCC cells [ASAP1 (hsa_circ_0085616). CircASAP1 controls the miR-326/miR-532-5p-MAPK1 pathway by sponging miR-326 and miR-532-5p expression by the ceRNA mechanism. This promotes the proliferation and invasion of HCC cells. Recent studies have suggested low miR-198 expression in HCC compared to healthy liver parenchyma. A study by Tan et al. [Qin et al.  have shoCC cells . Qin et CC cells  used RNAn et al.  revealedThe c-Jun N-terminal kinase (JNK), also known as stress-activated protein kinase, is a part of the MAPK family . A few sIn the study by Deng et al. , the MAPP38 MAPK is the most important member of the MAPK family for regulating the inflammatory response. The p38 MAPK signaling pathway is triggered by physiological stress, lipopolysaccharide, osmotic stress, and UV irradiation. In liver cancer, miRNAs involved in the p38 MAPK signaling pathway regulate some biological behaviors of liver cancer cells.Some miRNAs regulate the behavior of HCC cells by a direct association with the p38 MAPK signaling pathway. Chu et al.  revealedFOXO1), which inhibits the release of proinflammatory cytokines and HCC progression. Together, these results indicate that several miRNAs regulate the expression of several molecules via the p38 MAPK signaling pathway and indirectly affect HCC progression.Some miRNAs affect HCC development by an indirect association with the p38 MAPK signaling pathways. According to Liu et al. , in HCC Based on these studies, it can be found that some microRNAs can participate in a separate MAPK pathway to affect HCC progression .In addition to the previously mentioned miRNAs, which target a specific MAPK signaling pathway, some miRNAs simultaneously target multiple MAPK signaling pathways and participate in the complex regulatory network of HCC development. Some of the miRNAs and their associated mechanisms are described as follows:Wang et al.  showed tAdditionally, some microRNAs associated with the MAPK signaling pathway regulate the occurrence and progression of liver cancer. These microRNAs also alter drug resistance, thereby affecting liver cancer treatment via multiple mechanisms or compounds. The first are the microRNAs associated with drug resistance. Dietrich et al.  used bioFurther, various compounds stimulate miRNA expression. Autotaxin, a secretory ectonucleotide enzyme, metabolizes lysophosphatidic choline to produce lysophosphatidic acid, which is distributed throughout the body. In addition to phospholipase activity, autotaxin, a phosphodiesterase, also influences inflammation and cancer progression ,60,61. KBased on these studies, it is evident that some microRNAs are simultaneously associated with multiple MAPK signaling pathways. This indicates that they do not affect a single pathway but have a holistic effect on the MAPK signaling network, thus regulating HCC development. It implies that miRNAs are closely related to the MAPK signaling network in HCC .NcRNAs longer than 200 nucleotides are arbitrarily characterized as lncRNAs . LncRNAsMAPK6 is abundantly expressed by HCC samples [MAPK6 promoter and recruits RNA polymerase II. This results in the activation of MAPK6 (ERK3) transcription, the enhancement of the MAPK signaling pathway, and the self-renewal of liver tumor-initiating cells via MAPK6 expression. Previous studies have shown that an increase in the expression of lncRNA HOXD-AS1 was observed in HCC cell lines/tissues, which facilitates the metastasis of HCC cells by various mechanisms [Most lncRNAs related to the ERK signaling pathway promote liver cancer. Jiang et al.  used Hep samples . Furtherchanisms ,87. A stchanisms  confirmechanisms . Moreovechanisms .In addition to the lncRNAs independently associated with the ERK signaling pathway, some lncRNAs associated with the p38 MAPK signaling pathway also affect the inflammatory response and metastasis in liver cancer.As mentioned previously, in relation to miRNAs, increased levels of lncRNA HULC in HCC attenuates NF-κB and p38 MAPK signaling pathways in TNF-treated ATDC5 cells by downregulating microRNA-101. This protects ATDC5 cells against TNF-α-induced inflammatory damage . FurtherDDIT3, DUSP4, and RAP1A and regulate the MAPK, p53, and HIF1α signaling pathways to promote HCC cell proliferation. A previous study has shown that RAP1A stimulates the ERK, p38 MAPK, and Notch signaling pathways, which induces the metastasis of ovarian cancer cells [Some lncRNAs are not solely associated with a specific MAPK signaling pathway in primary liver cancers but can be related to several MAPK signaling pathways for regulating the progression of liver cancer. Some lncRNAs regulate cell proliferation to influence HCC progression. A study by Xu et al.  reporteder cells . Therefoer cells  demonstrSome lncRNAs regulate apoptosis. A previous study has shown an increase in lncRNA HCG11 levels in HCC, which inhibits apoptosis and promotes HCC progression. LncRNA HCG11 mediates this effect by interacting with IGF2BP1, which interferes with the phosphorylation of the anti-apoptotic factors ERK, JNK, and p38 . The inhIn addition to microRNAs and lncRNAs, circRNAs associated with the MAPK signaling pathway also influence primary liver cancer. CircRNAs are produced by the back-splicing of protein-coding mRNAs or linear ncRNAs, connecting the upstream 3′ and downstream 5′ splice sites, forming a covalently closed continuous loop .Some circRNAs associated with the MAPK pathway inhibit HCC. CircSETD3 (hsa_circRNA_0000567/hsa_circRNA_101436) is a novel HCC tumor suppressor. CircSETD3 sponges miR-421 via the ceRNA mechanism, which prevents the miR-421-induced degradation of MAPK14 p38 MAPK), thus inhibiting HCC progress via the circSETD3/miR-421/MAPK14 pathway . Wang et MAPK, thIn addition to circRNA, small nucleolar RNA (snoRNA) also associated with the MAPK signaling pathway influences HCC. SnoRNA is a class of extensively studied small ncRNAs and participates in the chemical modification of transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), and small nuclear RNAs (snRNAs) to improve RNA folding . Xu et aOne of the most extensively studied lncRNAs is the metastasis-associated lung adenocarcinoma transcript 1(MALAT1) . The nucFurther, vault RNA (vtRNA) is a novel family of short non-coding RNAs . VtRNA iTogether, these results suggest that other non-coding RNAs contribute to the development of liver cancer via the MAPK signaling pathway .Despite the advancement in therapeutics, the overall survival rates of HCC patients are still low. This could mainly be due to a lack of early diagnosis and the high heterogeneity of tumors. Hence, it is crucial to develop new molecular targets. A close association between the MAPK signaling pathway and liver cancer has been established, and ncRNA also plays a significant role in the progression of liver cancer. Hence, the association between ncRNA, the MAPK signaling pathway, and liver cancer should be extensively analyzed. In this review, we summarized the molecular mechanisms of microRNA, lncRNA, and other ncRNAs in the occurrence and development of primary liver cancer associated with the MAPK signaling pathway. Our analysis showed that some ncRNAs are associated with a specific MAPK signaling pathway, which participates in the development of liver cancer by regulating the expression or phosphorylation of specific proteins. Further, some ncRNAs are extensively involved in several MAPK signaling pathways, regulating the expression of proteins associated with the ERK, p38 MAPK, and JNK signaling pathways, which affect the progression of liver cancer. Furthermore, some ncRNAs are also stimulated by some compounds that affect the MAPK signaling pathway or are involved in drug resistance by regulating the MAPK signaling pathway.With the rapid development of sequencing and bioinformatics technologies, various ncRNAs are constantly being discovered, some of which play a significant role in cancer development. Hence, drugs targeting these ncRNAs can be used for treating cancer. For example, the first miRNA drug, miR-122 antagonist Miravirsen, has entered phase II clinical trials . TherefoAdditionally, significant developments have been made in exploring ncRNA in recent years. A few studies have demonstrated that some ncRNAs can be translated. As mentioned earlier, the peptide SMIM30 encoded by lncRNA LINC00998 regulates cell proliferation and migration to promote the progression of HCC. SMIM30 influences HCC progression by binding to SRC/YES1, which drives its phosphorylation and membrane anchoring, promoting the downstream ERK1/2 and p38 MAPK signaling pathways . In addiIL1R and TGFBR, which are upstream of the MAPK signaling pathway, share a common grade junction pathway (p38 MAPKs-MEF2C), which induces the proliferation, differentiation and apoptosis of cells [NcRNAs target the MAPK signaling pathway and upstream and downstream target molecules in various cancers. Several upstream cytokines and membrane receptors activate the MAPK signaling pathway. In gastric cancer, miR-143 indirectly downregulates the expression of the human epidermal growth factor receptor (HER2), which is an upstream molecule of KRAS, by silencing DEAD/H-box RNA helicase 6 (DDX6) . In breaof cells . In addiof cells . MiR-155of cells . In maliof cells .The upstream RTKs receptors of MAPKs play a critical role in the activation of signaling pathways, including EGFR, hepatocyte growth factor receptor , Vascular Endothelial Growth Factor receptor (VEGFR), platelet derived growth factor receptor (PDGFR), etc. In HCC, these receptors are also regulated by some microRNAs. MiR-206 inhibits the expansion of liver cancer stem cells and HCC cell dedifferentiation by targeting the EGFR signaling pathway . The miRCIP1/WAF1 pathway, which arrests the cell cycle at the G1 phase [The MAPK signaling pathway activates the downstream target genes and induces transcription of various genes, including cell-cycle-associated genes such as CDK4/6. These genes are also regulated by microRNAs. In Osteosarcoma (OS) cells, miR-338-3p acts as a tumor suppressor by targeting runt-related transcription factor 2 (RUNX2) and CDK4, which inhibits the MAPK pathway . In bladG1 phase . These rAs mentioned earlier, miR-622  and miR-In 2010, Settleman et al.  detectedBRAF) inhibitor for treating BRAF-V600K-mutant metastatic melanoma [A combination of inhibitors has been used to address the issue of drug resistance in cancer therapeutics. EGFR inhibitors are often utilized for the treatment of EGFR-mutant lung cancer patients; however, acquired drug resistance has reduced its efficacy. A study has shown that the combined inhibition of Aurora B kinase and EGFR prevents and eliminates the resistance to EGFR inhibitors in lung cancer by promoting BIM and PUMA-mediated apoptosis . Severalmelanoma . As a MEmelanoma . Howevermelanoma ,136. SCHmelanoma ,138,139.melanoma . The commelanoma . This evTogether, these results suggest that the regulation of the MAPK signaling pathway and its upstream as well as downstream molecules by non-coding RNAs plays a vital role in the development, diagnosis and treatment of liver cancer. In this review, we only explored the non-coding RNAs associated with the MAPK signaling pathways in liver cancer. A molecular network is composed of multiple interrelated signaling pathways in the progression of HCC. Some non-coding RNAs simultaneously affect multiple signaling pathways, which play a more significant role in the occurrence and development of liver cancer. MAPK inhibitors are crucial for cancer therapeutics. Further, a combination of inhibitors targeting ncRNA and MAPK pathways could be a promising candidate in cancer therapeutics, especially in drug resistance .


Nettle is kind of new feed resources and benefit for animal production. However, a few studies observed that quality of nettle silage was poor under naturally fermentation. Consider of microbial activity was the mainly factors for fermentation characteristics of silage.Thus, the present study investigated the potential factors causing nettle silage failure through metabolome and bacterial community composition analyses during ensiling.Enterococcus, Weissella, and Pediococcus were the dominant bacteria , with stable trends, whereas Lactococcus and Enterobacter relative abundance decreased significantly over time . Lactobacillus relative abundance was <1% during the entire ensiling period, and malic acid metabolic pathway was the most important pathway. Enterococcus, Pediococcus, and Weissella were negatively correlated with malic acid, with Lactobacillus displaying an opposite trend.During ensiling, the pH was >6.22, and water-soluble carbohydrate and organic acid contents stabilized after 7 d. At the genus level, Lactobacillus activity was the lowest among lactic acid bacteria (LAB) during ensiling, which is the main reason for nettle ensiling failure, and attributable to a low capacity to compete for fermentation substrates such as malic acid against other LAB during ensiling. Additionally, anti-bacteria activity of nettle probably inhibited Enterobacter activity during ensiling. Present study probably given a solution for improve nettle silage quality through addition with malic acid.The results suggested that  In recent years, the demand for feed resources has increased in the wake of livestock feed factory development, and one potential solution is exploitation of non-conventional feed sources . NettlesSeveral studies have reported that nettle as feed can improve animal performance and production, for example, by enhancing rumen health in lactating cows, promoting chicken growth, improving growth and carcass quality, and enhancing cow milk quality and quantity . AdditioLactobacillus plantarum at a sufficient fermentation substrate level (6.68% DM of WSC) still fails to facilitate ensiling. In addition to the high moisture content in nettle, its antimicrobial activity is a potential critical factor causing its poor ensiling properties.Nettle leaf has high protein [30% of dry matter (DM)], fiber (10%), and fat (4%) contents, and the biologically-active compounds include terpenoids, carotenoids, vitamins, cis-9,12-linoleic, α-linolenic acids, and polyphenolic compounds . HoweverEnterococcus, Enterobacter, Bacillus, Lactobacillus, etc., at the genus level, in dose dependent manners (Enterococcus and Lactobacillus are the most important lactic acid bacteria (LAB) observed during ensiling  was harvested on 10th September 2020 from the wild in Shawan County, central mountains of Tianshan, Xinjiang, China . The nettle was left to wilt to approximately 300 g/kg fresh weight. The samples were then chopped into 2 cm stalks using a forage cutter. After manual mixing, approximately 1.0-kg samples were packed into polyethylene plastic bags equipped with a one-way air extraction valve (23 cm × 30 cm). The bags were sealed using a vacuum sealer. There were five fermentation period treatments , with five replicates in each treatment, yielding a total of 25 bags of samples, which were stored at 24°C.Nettle (200 g silage samples (five replicates for each fermentation period treatment) were dried at 65°C for 48 h and ground to pass through a 1.0-mm sieve, and then used to determine DM content. Total nitrogen (TN) was determined using an automatic Kjeldahl nitrogen analyzer , and cured protein was calculated according to the method of the Association of Official Agricultural Chemists (AOAC). WSC content was determined according to 4-N) and organic acid concentrations, according to 2HPO4  and methanol were used for mobile phase A and B, respectively, with a flow rate of 0.6 ml⋅min–1, with an injection volume of 20 μL and oven temperature of 50°C. Following gradient profile: 0 min, 100% A; 5–40 min, 98% A.Fresh silage samples (20 g), with three replicates for each fermentation period treatment, were used to analyze fermentation characteristics. After using four layers of cheesecloth to filter the water-silage mixture (1:9 for v/v), the pH was measured using a portable pH meter , and the supernatant was collected for use in analysis of ammonium-N . Primers  targeting the V3–V4 regions of 16S rDNA were used to conduct PCR amplification. The amplicons were extracted, purified, and the raw sequences analyzed, according to (PRJNA859630).Total DNA in each sample (five replicates for each fermentation period treatment) was extracted using a commercial DNA Kit  was performed using the Vegan v2.5-3 package in R, based on Bray–Curtis dissimilarity . Significant differences between treatments were determined using Tukey’s test at milarity . FurtherThe WSC content in wilted nettle was 6.18% DM . AccordiR = 0.7309, P = 0.001).As shown in As shown in Enterococcus was dominant during the entire ensiling process, with a relative abundance of 30.06–39.39%, followed by Weissella (17.29–23.34%), Enterobacter (3.86–24.1%), Lactococcus (5.68–13.96%), and Pediococcus (3.13–7.22%). In addition, the relative abundances of Enterococcus, Pediococcus, and Weissella stabilized during ensiling, whereas the relative abundances of Lactococcus and Enterobacter decreased in the course of ensiling (p < 0.05). Spherical LAB, such as Lactococcus, Pediococcus, and Enterococcus, exhibited greater activity during the early stages of LAB fermentation during ensiling, but were replaced by rod-shaped LAB, such as Lactobacillus, which have higher acid tolerance than spherical LAB  since they decrease the activity of hexokinase, acetate kinase, and glycolytic enzyme systems, and destroy the cell membrane structure; however, such interference occurs only when the pH is <5.As shown in ical LAB . In the Lactococcus abundance  is in the 7.5–125 μg/ml range  when compared with the inhibitory capacity of some antibiotics, such as erythromycin, ampicillin, ciprofloxacin, and gentamicin (MIC = 0.078–5 μg/ml)  . In addi quality , such efus study . TherefoIn total, 537 metabolites were detected in the present study. Among them, 91 metabolites were annotated based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, as shown in During nettle ensiling, the most active metabolic pathways were the arginine biosynthesis pathway, protein digestion and absorption pathway, aminoacyl-tRNA biosynthesis pathway, and central carbon metabolism in cancer pathway . In addiEnterococcus, Lactobacillus, Leuconostoc, and Weissella, and possess the ADI pathways  to communicate with each other via autoinducer-2 (AI-2), and AI-2 biosynthesis occurs sm cycle . Bacteritabolism . The resompounds . The resFour fatty acyls, including hydroxyoctadecadienoic acid (HODE), 9,10-dihydroxyoctadec-12-enoic acid (DHOME), derivative of DHOME (DiHOME), and prenyl glucoside, were involved in different metabolic pathways only during the early stage of ensiling. HODE, DHOME, and DiHOME were up-regulated whereas prenyl glucoside was down-regulated during the early stage of ensiling. HODE, DHOME, and DiHOME participate in the caryophyllene epoxide, perillyl alcohol, and valtrate biosynthesis pathways, respectively, during ensiling. Caryophyllene epoxide was down-regulated only during early stage of ensiling, perillyl alcohol was down-regulated during the middle and late stages of ensiling, whereas valtrate was down-regulated during the middle stage of ensiling but up-regulated during the late stage of ensiling. Valtrate is a small molecule anticancer agent that inhibits nuclear export signals of cells, and is extracted from plants in the family Valerianaceae . The incEnterococcus, Weissella, Lactococcus, and Pediococcus, which belong to LAB, had a total relative abundance of 65.11–69.13%. Consequently, malic acid was fermented mainly by LAB during the early stage of ensiling. So far, no researcher has been reported glutaric acid in silage. Most studies focus on glutaric acid production via microbial metabolic engineering approaches, via four metabolic pathways. Pseudomonas putida is the only microbe with the natural ability to produce glutaric acid through the aminovalerate pathway via the degradation of L-lysine -L-lysine, which is in involved in lysine degradation pathways, according to the KEGG database, was detected. The results indicate that some Pseudomonas strains potentially produce glutaric acid during nettle ensiling.Malic acid and gluratic acid are organic acids that participate in different metabolic pathways. In the present study, malic acid was down-regulated during the early stage of ensiling whereas glutaric acid was up-regulated during the entire ensiling process. Most LAB can convert malate into lactate through malolactic fermentation to directly produce lactate, or pyruvate, as an intermediate compound . During L-lysine . In the Enterobacter and Lactococcus were positively correlated with methionine . The AI-2-dependent bio-film production via the methionine catabolism cycle enhances stress resistance in E. coli . Lactococcus reportedly possesses the ADI pathway, which could produce citrulline through the hydrolysis of arginine . Malic acid had a 23.4–37.5% inhibition rate against AI-2 activity in E. coli, in a dose-dependent manner, via penetration of the bacterial cell wall and alteration of microbial cytoplasmic pH  . During food fermentation, the competitiveness of strictly heterofermentative LAB depends on the use of external electron acceptors . Six Lactobacillus strains have been reported to possess mle and mleS genes, which encode malic and malolactic enzymes, respectively  . Enteroctabolism . In the cceptors . Filanniectively . Similarc enzyme .L. plantarum growth rate by 70%, in turn increasing biomass yield of the bacteria by 25.18%, in continuous culture in a bacterial growth medium . During ensiling, The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in this article/RH: conceptualization, methodology, and writing—review and editing. FZ: resources, validation, supervision, and review and editing. YCC and YXC: resources and investigation. CM: resources and validation. SJ: resources. All authors contributed to the article and approved the submitted version.


Cancer in young women is a major health problem in the Middle Eastern and North African population. We explored the awareness, barriers and practice of Arab oncologists towards oncofertility.Oncologists from Arab countries treating female cancer patients were invited to complete a 30-item web-based questionnaire that explores oncologists’ demographics, available techniques and barriers to oncofertility.170 oncologists working in 9 different Arab countries responded to the questionnaire. Among the responders, 89 (52.4%) were from Egypt and the central region, 60 (35.3%) were from North Africa and 21 (12.4%) were from the Gulf region.While most participants considered a dedicated training ‘necessary’, only 43 oncologists (25.3%) received a formal training. Only 17 participants (10%) had a fertility clinic in their centre, 44 (25.9%) and 13 (7.6%) had to refer patients to other centres or other cities, respectively. A total of 96 oncologists (56.5%) did not have access to a fertility preservation service.Out of 147 responders, 79 (53.7%) offered fertility preservation only in patients presenting with early disease and 38 (25.9%) did not offer fertility preservation. In terms of proposed strategies, 50 responders (29.4%) offered embryo cryopreservation, 79 (46.5%) oocyte cryopreservation and 48 (28.2%) ovarian tissue cryopreservation.A large gap exists between international clinical practice guidelines and current practices of fertility preservation in Arab countries. Barriers to optimum service delivery include the lack of physician awareness/training, unavailability of some advanced techniques and a lack of dedicated fertility clinics within the cancer centres. The population distribution in Arab countries is much younger compared to the Western world: more than half 54%) of the population is younger than 25 years, 40% is from 25 to 60 years and only 6% is older than 60 years . This is% of the Fertility is one of the key aspects of quality of life for breast cancer patients diagnosed at childbearing age and is considered one of the pillars of good quality care of cancer when diagnosed at a young age . DespiteAccording to international guidelines, fertility preservation strategies should be discussed with all female patients treated for cancer at a young age. Oncologists should explore treatment-induced infertility risks and pay attention to the patient’s wish in having children, following treatment completion, implementing fertility preservation strategies as early as possible after diagnosis , 5.Several barriers are thought to prevent the access of young cancer patients to fertility counselling and fertility preservation strategies, including low awareness about the issues by the treating oncologist , 11. To Oncologists  from 15 Arab countries were invited to participate. The invited cohort was generated using a list of practicing physicians who registered in three oncology regional conferences. Participants completed a web-based survey to self-evaluate their knowledge, attitude and behaviour regarding oncofertility care in female cancer patients.A 30-item questionnaire (see Appendix) was prepared by two authors (LK and KO) independently; then, a consensus on the final list of questions was established. The questionnaire was designed to explore the oncologists’ demographics, availability of fertility preservation methods and barriers to fertility counselling (see Appendix). The link to the survey conducted via Google Forms was distributed to 320 oncologists  via email. The response time window was 30 days and a reminder email was sent to non-responders on day 20.A consent statement was added to be accepted by the participants before proceeding to the questionnaire.p-values below 0.05 were considered statistically significant. The raw data were exported to Microsoft Excel and the analyses were conducted using IBM SPSS statistics version 20.Descriptive analyses were conducted. Chi-square test was used for the correlations between categorical variables. All One hundred and seventy oncologists working in nine different Arab countries responded to the survey out of the invited 320 participants (response rate = 53.1%). The participants worked in different Arab regions, with 88 (51.8%) from Egypt, 60 (35.3%) from North Africa and 21 (12.4%) from the Gulf region. Median age was 35 years (range: 25–65 years) and 99 (58.2%) were female. The participants’ demographics are shown in The median duration of oncology experience was 9 years (range: 1–35 years). The majority (78.2%) were medical or clinical oncologists, and 77.7% worked in an academic environment. Interestingly, while almost all the participants (97.6%) considered a dedicated fertility preservation training as “necessary”, only 43 oncologists (25.3%) received a formal training, 11 (25.6%) of them were through a self-sought independent course and not within the oncology training curriculum. Out of 30 participants, 12 (40%) could describe the duration of the fertility course/training they received to be from 1 to 3 hours. Regarding their current practices, 89 of 170 participants 52.4%) declared that they refer to clinical practice guidelines about fertility preservation in cancer patients, the majority of which referred to US  and Euro% declareAlthough the majority (82.4%) of oncologists mentioned that they see more than 10 newly diagnosed young female cancer patient per year, only 17 (10%) had a fertility counselling clinic in their centre/hospital, 44 (25.9%) had to refer patients to other centre or even other cities  for fertility counselling, while 96 oncologists (56.5%) did not report the presence of a dedicated fertility preservation service. The physician who usually leads the fertility counseling was the treating oncologist (25.5%), a gynaecologist (54.1%) or both (20.4%). Only 30 out of 147 respondents (20.4%) offered fertility preservation to early and metastatic patients, 79 (53.7%) offered it to women presenting with early disease only, while 38 (25.9%) did not offer fertility preservation to any patient. Regarding the usually proposed fertility preservation strategy, 79 (46.5%) were able to offer oocyte cryopreservation, while 142 (83.5%) physicians could offer Leutinizing hormone releasing hormone (LHRH) agonist during chemotherapy to their patients. Embryo cryopreservation and ovarian tissue preservation were not widely adopted  .Several barriers were explored in our study. First, the fear of delaying the active treatment  for advanced stages of cancer was a major barrier. One hundred and thirty-nine physicians (81.8%) believed that – at least sometimes – the patients could get delayed as a result of referral to fertility clinics. This might be the reason why 53.7% of the physicians only refer patients with early disease and 25.9% do not offer the service to all patients. The second barrier is the technical and financial availability. As mentioned before, advanced techniques are not available at most centres. Of note, 140 physicians (82.4%) stated that LHRH agonist was the only modality financially covered by the conventional health insurance in their practice. Finally, a minority of the participating physicians reported facing relevant religious/cultural (11.2%) or legal barriers (15.3%) to providing fertility preservation techniques to young female cancer patients.We conducted a survey-based study to assess the awareness and practices of Arab oncologists towards oncofertility. Our study demonstrated some striking findings. First, around three-quarters of the participating oncologists did not receive any sort of training for fertility preservation in young cancer patients. Second, only a minority of oncologists in the Arab world were able to offer an integrated fertility preservation services for female cancer patients. LHRH agonist given during chemotherapy for ovarian function preservation was the only available method for the majority (82.4%) of the participating oncologists. More established fertility preservation techniques are rarely available/affordable.Our study highlights important gaps in a different approach from other previous existing studies. We invited physicians from different disciplines of cancer care and various Arab countries to gain a wider view of their current attitudes in the oncofertility field. In addition, we explored the practices regarding different types of cancers in young women . According to prior studies, several barriers exist in the way adequate oncofertility counselling is managed . The phyWhile most guidelines suggest that oocyte and embryo cryopreservation are the preferred first-line FP options for young female patients with cancer , 7, 15, et al [Several studies have investigated similar gaps worldwide. A survey-based study carried out among 273 oncologists attending the 2016 BCY and the 2017 St Gallen conferences found that 37% of the participants had never refered to existing guidelines regarding FP in breast cancer and that a significant proportion had major misconceptions regarding the issue . A FrencRegarding the profile of our participants, the majority were young oncologists and 58.2% were female. A Japanese study that included 434 oncologists treating breast cancer patients found that young female oncologists were more likely to refer patients to a fertility specialist/centre . We did In the Japanese study, oncologists with higher knowledge scores and those who worked in a multidisciplinary environment were more likely to offer FP. This highlights the importance of establishing a dedicated FP training programme as a part of the oncology residency/fellowship training programmes. Similar to our finding, the risk of disease recurrence/progression and fear of treatment delay were major barriers to offer fertility preservation with breast cancer patients .Few studies have assessed the gaps in Arab countries , 19. A sOur study is, however, limited by not being able to cover all the Arab countries and all the different practice types. Moreover, and due to the survey-based nature of the study, we were not able to assess the needs of healthcare systems in Arab countries in full depth.In conclusion, there is a large gap between the international clinical practice guidelines and the current practices regarding fertility preservation in female cancer patients. The barriers to optimum service delivery in Arab countries include inadequate physician awareness/training and the unavailability of some advanced fertility preservation techniques, in addition to some financial constraints. Adding a dedicated FP training to oncology residency and fellowships, encouraging multidisciplinary practice and collaboration and offering financial coverage of FP techniques are essential to advance oncofertility worldwide, including in Arab countries.Loay Kassem received honoraria from Roche, Lilly, Novartis, Pfizer and Sandos; all outside the scope of the submitted work.Marwan Tolba, Nawfel Mellas and Karima Oualla have no conflicts of interest to disclose.Matteo Lambertini is a consultant for Roche, AstraZeneca, Novartis and Lilly, and received honoraria from Roche, Lilly, Novartis, Pfizer, Sandoz and Takeda; all outside the scope of the submitted work.All participating physicians provided informed consent  before participation. No identifiable patients or physicians’ information was included in the study.The study did not receive any external funding.


The dynamics of optical nonlinearity in the presence of gain and feedback can be complex leading to chaos in certain regimes. Temporal, spectral, spatial, or polarization instability of optical fields can emerge from chaotic response of an optical  The 7.Nonlinear optical media are being investigated for the generation of quantum optical states in both continuous variable and discrete variable regimes. The 10. The 11, where temporal and spatial instabilities have been observed for transverse modes. An effective resonator can be created from counter-propagating pumps. In this way, the Bragg grating induced by the standing-wave pump15, supports amplified multi-wave mixing (AMWM) for distinct spatial modes. Above certain gain values, temporal and spatial instabilities of the individual Stokes and anti-Stokes modes can be observed10. Although the dynamic and dependency on experimental parameters are complex, the mathematical derivation of instability against transverse perturbations is straightforward16. Nevertheless, the nature of the dynamic and static instabilities, and multimode correlations that appear, despite the intensity instability of individual modes, is not well understood. The sensitivity of the process to the initial conditions, size, power etc, can enable investigation of spatial and spectral phase transitions19 for sensing and optical switching applications in both classical and, in principle, quantum regimes.It is known that nonlinear optical media inside resonators can exhibit instabilities in space and time as a manifestation of chaotic response of nonlinearity in presence of feedback. Above some gain threshold, the feedback provided by the resonator can lead to a chaotic response or bi-stabilities in some degrees of freedom20 of Stokes and anti-Stokes light emerges with signatures of instability. The instability can be static  or dynamic (oscillating patterns in time). Although the scattering patterns are instable, the Stokes and anti-Stokes modes can still be correlated. Two-mode correlation of this kind has been shown using AMWM process in an Na vapor cell11. Here, we study amplified four-wave mixing between multiple spatial modes (or AMWM) in Rb vapor and characterize the correlation using single-photon Electron-multiplying CCD (EMCCD) cameras. The camera enables us to simultaneously study correlation, noise and instability in the counter-propagating modes.A counter-propagating pump creates an atomic Bragg reflector while the multi-wave mixing process is primarily amplified for atoms with zero velocity. As the result, Stokes and anti-Stokes photons are generated symmetrically with respect to the pump axis see Fig. . We obseA schematic of the experimental setup is illustrated in Fig. e/s and exposure time as low as 10 The ring-shaped counter-propagating scattered light was collected from the two ends of the atomic vapor cell and then detected using two areas of one EMCCD camera. To have the detection area independent of the propagation length, an image of the scattered light was created in the Fourier plane. The EMCCD camera had single-photon sensitivity with an active area of 128 × 128 pixels, dark noise 0.02 20.For long camera exposure times, the scattered ring intensity appeared continuous in space, but for short exposure times a periodic pattern in the scattered intensity was observed. We associated the fringes in the scattered intensity captured at low exposure times to the spatial instability of AMWMWe observed that the ring-shaped scattering appeared around three regions of pump detunings divided by the two optical transitions of Rb. The scattering signal could not be observed outside these regions. At certain pump detunings, the two Raman transitions (between pump and Stokes photons and pump and anti-Stokes photons) interfere and the amplification of the anti-Stokes photons occurs. We observed that as the pump frequency was tuned away from these resonances, the scattering intensity decreased until the signal was no longer visible. In what follows, we set the pump frequency to be red-detuned  induces an angular momentum on the standing wave24. This angular momentum is transferred to the scattered light resulting in an interference pattern along the ring. We observe a linear dependency of the spatial frequency on the pump power  around the ring. The spatial instability can be modeled, to the first order, using Gaussian beam propagation and interference considering a phase conjugate mirror$$\chi ^{}$$\end{dThe refractive index modulation, due to the standing-wave pump, coherently splits the scattering light propagating in opposite directions (i.e. the Bragg effect). Along with co- and counter-propagating four-wave mixing directions, this gives rise to the observation of two-mode and four-mode correlations in our system. Below, we first calculate the normalized intensity-difference noise of the AMWM signal for multiple spatial modes and compare it with the intensity-sum noise of the same two modes.We evaluated the noise in the intensity difference between different regions of the counter-propagating rings. We calculated the intensity-difference noise (or variance) normalized by intensity-sum noise, as a function of the relative angles between the rings. Figure The angular intensity correlation between counter-propagating rings is plotted in Fig. Figure Figure To estimate the number of distinct spatial modes that show relative noise reduction with the counter-propagating modes, we calculate the intensity noise for a segment as small as a camera pixel. As shown in the inset of Fig. 11. When Stokes (anti-Stokes) photons are highly absorbed, only anti-Stokes (Stokes) photons will survive. This will lead to loss of correlation for photons propagating in the same direction while counter-propagating anti-Stokes (Stokes) photons maintain their correlations. Figure 11 and the alignment mismatch of the counter-propagating pump beams.The correlation between two co-propagating or counter-propagating AMWM signals can be calculated using covariance normalized to variance values, 5.We also evaluate four-point correlations by calculating the 4th-order correlation function, 28 where an incident probe is amplified and a correlated conjugate is created. Also, the amplified nature of the multi-wave mixing process enables observation of bright Stokes and anti-Stokes signals with lower pump powers compared to the spontaneous four-wave mixing process30. We also observed that the relative noise exponentially decreases with the pump frequency within a range of pump detunings , we split the laser before the cell and measure the intensity-difference noise of the coherent laser light for varying power. We also measured the intensity-difference noise for the AMWM signal after the cell. The results are shown in Fig. see Fig. b. At lar31. To model the effect of the external broadening on re-absorption, we write Maxwell-Bloch equations and solve for Stokes and anti-Stokes field in presence of a two-photon detuning gradient31, z. Considering a pump light detuning much larger than the excited state linewidth, i.e. 31.Another factor limiting the degree of correlation is single-photon or two-photon re-absorption of Stokes or anti-Stokes photons. This effect can be reduced applying an atomic frequency gradientTo introduce the atomic frequency gradient, we apply a linearly varying Zeeman broadening using a solenoid. Figure To extract the maximum correlations between different spatial modes, the exposure time of the camera should be shorter than the coherence time or the inverse of the Raman scattering bandwidth, whichever is smaller. The photons arriving at different times within a single integration time (a single camera frame) are not expected to have correlations and they reduce the strength of noise reduction in the measurement. The lack of effective noise subtraction is evidenced by quadratic dependency of both intensity-sum and intensity-difference noise as a function of mean intensity. Therefore, the correlation measurement described above is an underestimation of the maximum correlations that exists in the system. The relatively large exposure time of the camera cannot be compensated for by using lower pump powers to reduce the scattering rate. Below the threshold pump power, the amplification could not be observed, and also at low scattering rates, the electronic noise of the camera overcomes the shot noise. Also, a camera with higher spatial resolution images will allow us to more accurately determine the maximum correlation and mode capacity of the source. By using two similar cameras (one for each ring) or one camera with higher temporal and spatial resolutions, larger correlations can be extracted from the images.17, multimode optical switching18, quantum imaging33, and quantum communications34 using room-temperature sources. For example, by reflecting one beam from an object and detecting both correlated beams on a CCD camera, the intensity correlations can be used to measure change in the reflectivity beyond the state-of-the-art classical devices35.The study reported here sheds light on some complex mode dynamics, transition to instability, and correlations in driven dispersive nonlinear resonators. The multimode and spatially-distinct nature of the correlations in the AMWM studied here can also find applications in study of phase transition


In PD patients, the extent of bradykinesia and rigidity correlated with VSA , while no correlation between axial gait symptoms or tremor and vowel articulation was detected. Vowel articulation impairment represents an early prodromal symptom in the disease process of synucleinopathy. Acoustic assessment of vowel articulation may provide a surrogate marker of synucleinopathy in scenarios where a single robust feature to monitor the dysarthria progression is needed.Imprecise vowels represent a common deficit associated with hypokinetic dysarthria resulting from a reduced articulatory range of motion in Parkinson’s disease (PD). It is not yet unknown whether the vowel articulation impairment is already evident in the prodromal stages of synucleinopathy. We aimed to assess whether vowel articulation abnormalities are present in isolated rapid eye movement sleep behaviour disorder (iRBD) and early-stage PD. A total of 180 male participants, including 60 iRBD, 60 de-novo PD and 60 age-matched healthy controls performed reading of a standardized passage. The first and second formant frequencies of the corner vowels /a/, /i/, and /u/ extracted from predefined words, were utilized to construct articulatory-acoustic measures of Vowel Space Area (VSA) and Vowel Articulation Index (VAI). Compared to controls, VSA was smaller in both iRBD ( Considering the development of Parkinson’s disease-modifying treatment3, a multicentre study including 1280 iRBD patients identified quantitative fine motor skill testing as the strongest predictor for conversion4. Another study by Postuma et al. 5 revealed that voice and face akinesia represent the earliest prodromal motor manifestations in iRBD subjects preceding the onset of parkinsonism by a mean 9.8 years. This is likely a consequence of motor speech complexity and its sensitiveness to neural damage6.Isolated rapid eye movement sleep disorder (iRBD) is a parasomnia characterized by dream-enactment behavior and loss of physiologic muscle atonia during the rapid eye movement sleep phase. iRBD is considered a prodromal stage of neurodegeneration as more than 80% of diagnosed patients developed alfa-synuclein-aggregation disorders such as Parkinson’s disease (PD), Lewy body dementia, or multiple system atrophy7. Moreover, speech impairment is present in a majority of newly diagnosed PD patients9. Considering that patients with iRBD are at high risk of developing PD, the speech behavior assessment in iRBD is subjected to thorough investigation. Recent multilanguage research based on fully automated analysis of seven distinctive speech dimensions of hypokinetic dysarthria10, including harsh voice, slow sequential motion rates, imprecise consonants, monoloudness, monopitch, prolonged pauses, and articulation rate performed on 150 iRBD patients revealed that only monopitch was able to significantly differentiate iRBD patients from controls11. Interestingly, monopitch was found in iRBD subjects with impaired olfactory function before the nigrostriatal dopaminergic transmission is affected12, that is, in Braak stage 2 before the substantia nigra is affected by synucleinopathy13. Among monopitch, vowel articulation impairment represents one of the core deficits contributing to dysarthric speech, as it reflects the range of articulatory movements and strongly correlates with overall intelligibility16. The potential of imprecise vowel articulation to serve as an early biomarker can also be supported by a previous pilot study where deficits in vowel articulation were detected in a small sample of 20 patients with de novo PD17. However, potential changes of vowel articulation in iRBD have never been investigated. Also, no previous research independently related articulation impairment to other essential prodromal features of synucleinopathy, such as olfactory dysfunction.Hypokinetic dysarthria of PD, which is mainly characterised by articulatory, phonatory and prosodic alterations, occurs in up to 90% of patients over the course of the diseaseThe purpose of this study is to investigate vowel articulation in iRBD and early-stage PD patients compared to healthy controls in order (i) to verify the prospect of using measurement of vowel articulation as a biomarker for the detection of prodromal PD and (ii) to investigate the relationship between articulation measures and the degree of motor and smell dysfunction.p = 0.001, η2 = 0.08]  and PD (p = 0.001) compared to HC individuals. In addition, group differences were also detected for Vowel Articulation Index (VAI) , as the PD group manifested significantly smaller VAI (p = 0.002) compared to HC group. Slight vowel duration differentiation was also observed across groups   groups are summarized in Table 94] Fig. . In addi40] Fig. . No signr = −0.29, p = 0.03) and VAI . In addition, bradykinesia and rigidity subscore in PD patients showed negative correlation with VSA  and VAI  while neither correlation between postural instability and gait difficulty (PIGD) subscore and VSA  or VAI  nor between tremor subscore and VSA  or VAI  was detected  total in PD patients showed negative correlation with VSA . No other significant correlations were found between vowel articulation parameters and clinical scales in PD or iRBD.Regarding brain imaging, the putamen binding ratio in iRBD showed positive correlation with VSA 31 whereas the tendency toward a slower speech rate may be theoretically attributed to the degeneration of non-dopaminergic pathways8.Previous studies assumed that speaking rate may influence vowel articulation performance in dysarthrias34. Anatomical dispositions of the vocal tract reflect sexual dimorphism36, and we therefore cannot exclude a potential sex effect on our results. On the other hand, no sex-specific speech dysfunction in de-novo PD was found9. The findings of current study are based on a cross-sectional design. Future research is needed to estimate the sensitivity of vowel articulation features in long-term follow-up and in predicting phenoconversion from iRBD to established parkinsonism.This study has certain limitations. We enrolled exclusively male participants primarily because of the strong predominance of male subjects within iRBD patientsIn conclusion, the vowel articulation deficits in male subjects significantly differentiated both iRBD and de-novo PD patients from controls. Future studies should elaborate our findings in the female population, and vowel articulation analysis might then have the potential to serve as a speech biomarker indicating the prodromal stage of synucleinopathy. Introducing novel acoustic biomarkers enable to design a speech assessment battery allowing for valid and easy evaluation of a large number of subjects in clinical trials as speech assessment is inexpensive, noninvasive and digitally storable. Additionally, vowel articulation analysis may facilitate better speech phenotype classification of PD and has, thus, the potential utility for personalized medicine.From 2015 to 2021, a total of 180 male Czech participants, including a separate group of iRBD, PD, and HC participants, were recruited. Each participant provided written informed consent. The study received approval from the Ethics Committee of the General University Hospital in Prague, Czech Republic, and has been performed in accordance with the ethical standards established in the 1964 Declaration of Helsinki.37. None of the iRBD patients suffered a significant communication disorder or had a history of treatment with antiparkinsonian medication, nor were they taking any medication affecting sleep, cognition or motor function. The PD group consisted of 60 untreated drug-naïve male patients aged 61.8 (SD 11.6) years, fulfilling the Movement Disorder Society clinical diagnostic criteria for PD38. At the time of examination, none of the PD patients had a history of communication disorder unrelated to PD, nor were they taking any medication affecting sleep or cognition function.The iRBD group consisted of 60 male patients aged 65.6 (SD 7.1) years diagnosed according to the third edition of the International Classification of Sleep Disorders39. Symptom duration was assigned based on the self-reported occurrence of the first motor symptoms. To define specific movement disorder manifestations that may influence speech disorder, three subscores from the MDS-UPDRS III scale were calculated as follows: (i) PIGD subscore , (ii) Bradykinesia and Rigidity subscore  and Tremor subscore . Item 3.1 Speech of the MDS-UPDRS III was used for the perceptual description of overall dysarthria severity. In addition, patients were examined using the University of Pennsylvania Smell Identification Test (UPSIT)40, Montreal Cognitive Assessment and Scales for Outcomes in Parkinson’s Disease - Autonomic Dysfunction41. iRBD patients were categorized into two groups based on their olfactory function: iRBD group with preserved olfactory function (iRBD-POF) consisted of patients with UPSIT > 25 (i.e. normosmic to moderately microsmic) and the iRBD group with abnormal olfactory function (iRBD-AOF) of patients with UPSIT ≤ 25 (i.e. severely microsmic or anosmic). Patient clinical and demographic characteristics are summarised in Table Both iRBD and PD subjects were examined by a movement disorder specialist (P.D.) using the MDS-UPDRS IIIp = 0.15).The HC group consisted of 60 male volunteers of comparable aged 64.1 (SD 12.8) years, with no history of significant neurological or communication disorder. No group differences in age distribution were revealed between iRBD, PD and HC groups  placed approximately 5 cm from the subject’s lips. The speech signals were sampled at 48 kHz with 16–bit resolution. The recordings were obtained during one session with a speech specialist  who conveyed instructions to the participants. Each participant completed a series of speaking tasks, including reading twice a standardised passage composed of 80 words as part of a longer protocol lasting about 20 minutes. A reading passage . Using PRAAT, both the combined wideband spectrographic display and the power spectral density were used to determine the first (F1) and the second (F2) formant frequencies in Hz. A total of 30 vowels per passage were studied, including 10 occurrences of /a/, 10 occurrences of /i/, and 10 occurrences of /u/ -N-(3-fluoropropyl) nortropane  tracer according to European Association of Nuclear Medicine procedure guidelinesp < 0.05. All statistical analyses were performed in MATLAB .As the Kolmogorov-Smirnov test for independent samples showed that all acoustic variables were normally distributed, we used analysis of covariance (ANCOVA) with post hoc Bonferroni adjustment to assess group differences. Since there was wide variability in age among PD participants, age was considered as a covariate. The Pearson’s partial correlation analysis controlled for age was performed to test for significant relationships between the clinical and acoustic data. The level of significance was set to Further information on research design is available in the Reporting Summary