Isolated rapid eye movement sleep behavior disorder (IRBD) is a well‐established clinical risk factor for Lewy body diseases (LBDs), such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB).To elucidate whether serum microRNA (miRNA) deregulation in IRBD can antedate the diagnosis of LBD by performing a longitudinal study in different progression stages of IRBD before and after LBD diagnosis and assessing the predictive performance of differentially expressed miRNAs by machine learning–based modeling.Using genome‐wide miRNA analysis and real‐time quantitative polymerase chain reaction validation, we assessed serum miRNA profiles from patients with IRBD stratified by dopamine transporter (DaT) single‐photon emission computed tomography into DaT‐negative IRBD (n = 17) and DaT‐positive IRBD (n = 21), IRBD phenoconverted into LBD (n = 13), and controls (n = 20). Longitudinally, we followed up the IRBD cohort by studying three time point serum samples over 26 months.P < 0.05). Age‐ and sex‐adjusted predictive modeling based on the 12 differentially expressed miRNA biosignatures discriminated IRBD and PD or DLB from controls with an area under the curve of 98% .We found sustained cross‐sectional and longitudinal deregulation of 12 miRNAs across the RBD continuum, including DaT‐negative IRBD, DaT‐positive IRBD, and LBD phenoconverted IRBD  (false discovery rate Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder SocietyBesides clinical diagnosis of IRBD or imaging markers such as DaT single‐photon emission computed tomography, specific miRNA biosignatures alone hold promise as progression biomarkers for patients with IRBD for predicting PD and DLB clinical outcomes. Further miRNA studies in other PD at‐risk populations, such as LRRK2 mutation asymptomatic carriers or hyposmic subjects, are warranted. © 2022 The Authors.  Rapid eye movement (REM) sleep behavior disorder (RBD) is characterized by the lack of REM sleep–related muscle atonia, disinhibited motor activity, and dream‐enacting behaviors. The isolated form of RBD (IRBD) represents the prodromal state of the synucleinopathies because, after 14 years of follow‐up, 91% of the patients with IRBD are diagnosed with Parkinson's disease (PD) and dementia with Lewy bodies (DLB), collectively known as Lewy body disorders (LBDs), and rarely with multiple system atrophy (MSA).MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by mRNA cleavage and translation repression.123I‐2β‐carbomethoxy‐3β‐(4‐iodophenyl)‐N‐(3‐fluoropropyl)‐nortropane as previously described.All subjects provided written informed consent, and the Ethics Committee of Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS)‐Hospital Clínic de Barcelona approved the study. The IRBD diagnosis required a history of dream‐enacting behaviors, audiovisual‐polysomnographic confirmation of excessive electromyographic activity in REM sleep, and absence of motor and cognitive impairment.g for 10 minutes at 4°C. Serum volumes of 2 mL were removed from the supernatant, aliquoted in polypropylene CryoTubes , and stored at −80°C. We mixed 200 μL serum from each sample with 2 μL of yeast transfer RNA (tRNA)  at a final concentration of 10 μg/mL as a carrier for nucleic acid precipitation. Following the manufacturer's instructions, total RNA enriched in miRNAs was isolated using the miRNeasy Serum/Plasma Kit . The RNA concentration was determined on a NanoDrop ND‐3300 fluorospectrometer (Thermo Scientific). We performed an electropherogram analysis using an Agilent Small RNA Kit in n = 22 random samples as quality control for 6‐ to 40‐bp miRNA‐enriched fractions and observed high‐quality miRNA enrichment for all studied samples , preserved it for 30 minutes at room temperature, and centrifuged at 1500les Fig. .https://www.thermofisher.com/). Serum miRNAs from individual subjects were hybridized onto the probe set separate arrays for 42 hours and scanned using an Affymetrix GeneChip Scanner 3000 7G following the manufacturer's instructions. Data files generated by the Affymetrix GeneChip Command Console were processed with the Expression Console software to determine the data quality. Expression raw data were analyzed using the Partek Genomic Suite v7.0 software (Partek Inc.) applying the robust multiarray average background correction model, which permits the relative comparison of miRNA abundance in different arrays. Only miRNAs with detection values greater than 2.4 arbitrary luminescence units in at least 50% of all samples, patients, and controls and a significant expression value above the background signal (P < 0.05) were considered as expressed in serum. In line with previous reports,t test, we applied the criteria of a combination of a fold‐change > |1.5| and P < 0.05 to select candidate miRNAs from the array for subsequent real‐time quantitative polymerase chain reaction (RT‐qPCR) validation  , thus validating findings.miRNA samples were reverse transcribed into cDNA and preamplified using the TaqMan Advanced miRNA cDNA Synthesis Kit  in a Veriti 96‐well Thermal Cycle (Applied Biosystems). cDNA preamplified products were quantified per duplicate using TaqMan Fast Advanced Master Mix  and TaqMan Advanced miRNA Assays  Table . ReactioAs Table , 10 wereAs Table . The remAs Table  and founTo predict each patient's probability of having the disease  versus being a control, we computed binary classification analyses using the caret framework in R, specifying age, sex, and RT‐qPCR expression values of the 12 validated serum DEmiRs as predictors. Multifold imputations of missing data were performed by the predictive mean matching method implemented in the mouse R package. Numerical variables were centered and normalized, resulting in a standard distribution of mean 0 and variance 1. The gradient boosting machine (GBM) algorithm in the gbm package (v.2.1.8) was employed to classify patients in the two groups (disease vs control), using leave‐one‐out cross‐validation to cut for potential overfitting. Variable selection was applied to each training dataset using the least absolute shrinkage and selection operator (Lasso) method in the glmnet package (v.2.0‐18) Table  so that https://ccb-compute2.cs.uni-saarland.de/mieaa2).To investigate the biological functions and localization of the identified DEmiR, we performed a restrictive overrepresented biological enrichment analysis using only the 12 DEmiRs that were deregulated at all longitudinal time points of both DaT‐negative and DaT‐positive IRBD and also in LBD . To this end, we used the GeneOntology option from the miEAA 2.0 software, applying gene annotations from the miRTarBase database as earlier describedP value <0.05  and three from DaT‐negative IRBD patients .We first profiled overall miRNA levels in serum samples from patients with IRBD and healthy control patients as an approach to screen global miRNA expression variation and identify specific candidate miRNAs for validation. Compared with control patients, by genome‐wide miRNA expression analysis, we found 116 candidate miRNAs in DaT‐negative IRBD patients and 50 miRNAs in DaT‐positive IRBD patients, which surpassed the fold‐change threshold greater than |1.5| and a 05 Table , Fig. S2Next, by RT‐qPCR, we quantified the relative expression levels of the selected miRNAs in DaT‐negative IRBD, DaT‐positive IRBD, and LBD phenoconverted patients (PD and DLB), all compared with healthy controls Table . Of thesSubsequently, we performed a follow‐up miRNA expression analysis by RT‐qPCR using longitudinal serums from DaT‐negative and DaT‐positive IRBD patients. Beyond baseline, we used two additional sera covering 26 months Table . CompareWith the goal of discerning patients with the disease  from controls (n = 20) and evaluating the discriminative potential of miRNAs, we performed predictive modeling based on the serum expression levels of the 12 experimentally validated DEmiRs identified in the cross‐sectional analysis, adjusted by age and sex. During leave‐one‐out cross‐validation, predictor features selected by Lasso in 100% of the training sets were age, sex, miR‐1227‐5p, miR‐425‐5p, and miR‐451a Table . The 12‐To further test the miRNA biosignature discriminative power for unbiased subject classification, we performed an unsupervised hierarchical clustering analysis using R. Consistent with the clinical status, we observed clear segregation into three main subclusters, including controls, patients with IRBD, and PD/DLB phenoconverted patients Fig. . Driven Lastly, we conducted a biological enrichment analysis to explore the functionality of the genes targeted by the miRNAs differentially expressed at all longitudinal time points. We found substantial deregulation at biological terms previously involved in clinically manifested PD, such as ubiquitin‐dependent protein degradation, inositol triphosphate signaling, apoptosis,We profiled serum miRNA levels in a Spanish IRBD cohort stratified into the seriated disease progression stages of DaT‐negative IRBD, DaT‐positive IRBD with a dopaminergic deficit,To the best of our knowledge, this is the first miRNA study in patients with IRBD characterized by DaT‐SPECT with longitudinal follow‐up, but two previous studies explored miRNAs in IRBD. A first study using candidate miRNAs reported the deregulation of miR‐19b‐3p in IRBD serum (n = 104) up to 5 years before α‐synucleinopathy diagnosis.In PD and DLB, developing disease prediction strategies ahead of the motor and cognitive manifestations is a challenge for the early neuroprotective intervention.37 were reported in PD serum; miR‐19b‐3p, miR‐29c‐3p, and miR‐361 in PD or early‐stage PD peripheral blood mononuclear cells10 in PD whole blood. Other biosignature miRNAs were described in the central nervous system (CNS), such as miR‐19b‐3p, miR‐22, miR‐29c,Based on our findings, an exciting interpretation is that the identified biosignature could hold potential as progression or phenoconversion biomarkers for patients within the LBD spectrum. Our miRNA findings are novel in IRBD but largely consistent with previous studies in manifest PD. Ten of the 12 biosignature miRNAs were earlier deregulated in different PD or DLB biospecimens. In LBD peripheral tissues, miR‐19b‐3p,Functionally, the target genes from the 12 longitudinal DEmiRs are involved in ubiquitin‐dependent protein degradation, inositol triphosphate signaling, apoptosis,Despite the promising identification of a novel miRNA biosignature that is informative of different disease progression stages in α‐synucleinopathies, our study has limitations. First, we performed a restrictive miRNA expression analysis filtering in only miRNAs validated by RT‐qPCR, but additional candidates could also be nominated for validation. Second, our miRNA analyses have not been corrected for IRBD medication, such as melatonin, which has been shown to interact with specific miRNAs in mice.In summary, we identified a serum miRNA biosignature that is informative in discriminating healthy controls, patients with IRBD, and patients with PD/DLB and holds potential as a disease progression biomarker. If validated, our findings may have implications for disease prediction strategies and early detection of α‐synucleinopathies such as PD and DLB. Further studies in other prodromal PD/DLB or PD/DLB at‐risk cohorts are warranted.Conception and design of the study: Conception , Organization , Execution ; Patient recruitment .Acquisition and analysis of data: Design , Execution , Interpretation ; Review and Critique .Drafting of the manuscript or figures: Writing of the first draft , Draft editing , Review and Critique .R.F.‐S. and M.E. received funding from The Michael J. Fox Foundation for Parkinson's Research (MJFF) (grant MJFF‐000858). R.F.‐S. was supported by the Instituto de Salud Carlos III (grant PI20/00659). M.E. received funding from the Instituto de Salud Carlos III (grant PI20/00259).FIG. S1 Quality control assessment of serum miRNA by electropherogram.Click here for additional data file.FIG. S2 Venn diagrams representing the number of candidate miRNAs from the genome‐wide miRNA analysis and DEmiR from the RT‐qPCR analysis in DaT‐negative IRBD, DaT‐positive IRBD and LBD compared to controls. IRBD, idiopathic rapid eye movement sleep behaviour disorder; DaT, DaT‐SPECT imaging; DaT(−), DaT‐negative IRBD patients; DaT(+), DaT‐positive IRBD patients; LBD, Lewy body disease (PD and DLB)Click here for additional data file.FIG. S3 Individual cross‐sectional and longitudinal ∆Ct values of DaT‐negative IRBD, DaT‐positive IRBD, LBD and controls as assessed by RT‐qPCR. DaT‐negative IRBDs are represented in blue, DaT‐positive IRBD in orange, LBD in purple and controls in green.Click here for additional data file.TABLE S1 Number of subjects treated for RBD and LBD and range of dose. IRBD, isolated rapid eye movement sleep behaviour disorder; DaT, DaT‐SPECT imaging; LBD, Lewy body disease (PD and LBD)TABLE S2: Timepoint intervals between longitudinal follow‐up sampling per each subject. Number of months between baseline sampling and timepoints 2 and 3 in DaT‐negative and DaT‐positive IRBD subjects. IRBD, isolated rapid eye movement sleep behaviour disorder; DaT, DaT‐SPECT imagingTABLE S3: Candidate differentially expressed miRNAs identified by genome‐wide microRNA expression analysis. (A) DaT‐negative IRBD versus controls. (B) DaT‐positive IRBD versus controls. IRBD, isolated rapid eye movement sleep behaviour disorder; DaT, DaT‐SPECT imaging; FC, Fold changeTABLE S4: Details of commercially available TaqMan Fast Advanced miRNA assays for assessment of miRNA expression levels by real‐time quantitative PCR (RT‐qPCR). (A) Assays used for RT‐qPCR assessment of miRNA levels. (B) Assays discarded due to poor RT‐qPCR amplificationTABLE S5: RT‐qPCR assessment of 10 selected miRNAs identified by genome‐wide miRNA expression analysis. IRBD, isolated rapid eye movement sleep behaviour disorder; RT‐qPCR, real‐time quantitative PCR; FC, fold change. (*) Differentially expressed miRNAs (DEmiR) from the array validated by RT‐qPCR with the same fold change direction and a statistically significant P‐valueTABLE S6: Technical validation of the real‐time quantitative PCR (RT‐qPCR) longitudinal analyses. Replication of the longitudinal RT‐qPCR assessment of miRNA expression levels in serum samples from DaT‐positive and DaT‐negative IRBD patients as compared to controls. IRBD, isolated rapid eye movement sleep behaviour disorder; DaT, DaT‐SPECT imaging; FC, fold change; adj. P, false discovery rate (FDR) multiple‐testing adjusted P‐valuesTABLE S7: Frequency of selection by LASSO of each miRNA included in machine‐learning analysis folds (N = 71).TABLE S8: Performance metrics achieved by machine‐learning analysis for detection of IRBD, PD and DLB (N = 71). (*) AUC values are adjusted by age and sex in the machine‐learning classifier. (**) P‐values obtained by Mann–Whitney U test. AUC, area under the curve; CI, confidence interval; Adj. P‐value, FDR multiple testing adjusted P‐value; IRBD, isolated rapid eye movement sleep behaviour disorderTABLE S9: RT‐qPCR assessment of miRNA expression levels in serum samples within the IRBD continuum. IRBD, isolated rapid eye movement sleep behaviour disorder; LBD, Lewy body disease (PD and DLB); DaT, DaT‐SPECT imaging; DaT(−) IRBD, DaT‐negative IRBD patients; DaT(+) IRBD, DaT‐positive IRBD patients; FC, fold change; Adj. P, FDR multiple‐test adjusted P‐valueClick here for additional data file.


The Wnt cascade is a primordial developmental signaling pathway that plays a myriad of essential functions throughout development and adult homeostasis in virtually all animal species. Aberrant Wnt activity is implicated in embryonic and tissue morphogenesis defects, and several diseases, most notably cancer. The role of Wnt signaling in mammary gland development and breast cancer initiation, maintenance, and progression is far from being completely understood and is rather shrouded in controversy. In this review, we dissect the fundamental role of Wnt signaling in mammary gland development and adult homeostasis and explore how defects in its tightly regulated and intricated molecular network are interlinked with cancer, with a focus on the breast. Deregulation of developmental signaling pathways is common in many cancer types, a feature that underpins the similarities between embryonic development and tumorigenesis . The WntDrosophila melanogaster by RP. Sharma, when performing mutagenesis screening of visual phenotypes. Subsequent studies identified several wingless-related factors that function as mediators of patterning during embryonic development (The wingless (wg) gene was discovered in 1973 in elopment .int1 (Wnt1), then found to be an ortholog of wg, generated mammary hyperplasia and breast tumors in mice  gene were discovered to be one cause of hereditary colon cancer. Soon after, the rcinomas .WNT1, the molecular complexity of the Wnt signaling cascade was progressively resolved, leading to the discovery of many components of this intricate pathway. Moreover, the intracellular responses triggered by Wnt ligands have been shown to branch into β-catenin-dependent signaling  and -independent signaling .Since the discovery of wg and int1). These growth factors are conserved throughout the animal kingdom and constitute a family of 19 known Wnt ligands in mammals, encoding for secreted glycoproteins whose function is to activate one or both branches of Wnt signaling in a paracrine or autocrine manner  adds a palmitoyl group before binding Evenness interrupted WNTless (Evi/WLS) to be shuttled to the plasma membrane via Golgi apparatus. PORCN acts as a rate-limiting enzyme in the Wnt secretory pathway as acylation of Wnt ligands is essential for their secretion  Figure .Secreted Wnt ligands directly interact with receptor complexes in receiving cells to activate canonical or non-canonical Wnt signaling. Receptor complexes, consisting of one of 10 members of the Frizzled (FZD) family of transmembrane receptors and co-receptor low-density lipoprotein receptor-related proteins 5,6 (LRP5/6), activate canonical Wnt signaling. On the other hand, receptor complexes consisting of a FZD receptor and Receptor Tyrosine Kinase-like orphan receptor 1/2 (ROR1/ROR2) or receptor-like tyrosine kinase (RYK) activate non-canonical Wnt signaling . family consists of four secreted proteins that contribute to Wnt signaling activation by synergizing with Wnt ligands. They act by binding members of the leucine-rich repeat-containing G-protein coupled receptors (LGR4-6), consequently inhibiting transmembrane E3 ubiquitin ligases, Rnf43 and Znrf3, involved in the recycling of FZD receptors . These t  and the Wnt/CaThe Wnt/PCP pathway is involved in establishing and regulating cell polarity, motility, and migration . Activat2+ pathway is critical in regulating cell adhesion, migration, and embryonic development , in this review, we mainly focus on the canonical Wnt signaling cascade due to the clearer general understanding of its underlying biology in health and disease and the high frequency of aberrant canonical Wnt activity in breast cancer.The mammary gland is a complex network of branched epithelial lobules and tubes which produce, collect, and transport milk to the nipple . This duDespite major differences between species (see below), our understanding of the molecular and histological processes underlying mammary gland development mainly stems from studies in model organisms, particularly murine models due to the limited availability of human pre-natal biological samples. From numerous studies in mice laboratory models, we know the very first morphogenic events during murine mammary gland development occur during embryonic day 10 with the appearance of the bilateral mammary lines between the limb buds. .Five pairs of mammary placodes arise from ectodermal cells in the mammary lines at E11 . By E13.Wnt10b mRNA is detected along the mammary line at E11.5 . Overexpression of PTHrP leads to improper differentiation of the ventral epidermis and ectopic expression of Lef1 and β-catenin, together with mesenchymal markers. Conversely, ablation of PTHrP expression disrupts transcriptional Wnt reporter activity  compartment has great implications for both development and disease, particularly in cancer. Roarty and colleagues described an intricate interplay between β-catenin dependent and independent signaling in the control of MaSC self-renewal and differentiation during mammary gland development and branching. Their work proposes a model in which β-catenin dependent signaling maintains the self-renewal of MaSCs in the terminal end bud (the leading edge of mammary gland branch extension). Conversely, Ror2 expression in the duct, being formed in the wake of the terminal end bud, leads to the differentiation of mammary epithelial cells within the basal and luminal breast lineage .+ cells in mammary epithelial cell cultures had significant stem cell activity  are elevated in this population , Basal-like (BLBC), and Normal-like (NLBC) breast cancers .Following this work, further classifications using gene signatures have been proposed. Using 706 cDNA probe elements, Sotiriou et al. identified six breast carcinomas groups: three luminal-like, one HER2-like, and two basal-like subtypes . LehmannWith the emergence of powerful technologies such as Next Generation Sequencing (NGS) platforms, more studies have provided extensive and detailed insight into breast cancer’s heterogeneous genomic and transcriptomic architecture for the discovery of new novel subtypes . A studyIn parallel, The Cancer Genome Atlas Network (TCGA) investigated breast cancer subtypes by incorporating data from multiple platforms, including, exosome sequencing, mRNA arrays, DNA methylation, genomic DNA copy number arrays, and more. They concluded that diverse genetic and epigenetic alterations converge phenotypically into four major breast tumor subgroups  as previously identified by Sørlie et al. . DespiteRoughly 70% of breast cancers are intrinsically dependent on steroid hormone signaling. The Luminal A and Luminal B breast cancer subtypes are positive for ER and/or PR . PatientFifteen to twenty percent of breast cancers overexpress the HER2 oncogene. HER2 is an orphan receptor whose activation leads to downstream induction of several pro-proliferative and pro-survival signaling cascades such as the mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and the phosphatidylinositol 4,5-biphosphate 3-kinase (PI3K) . HER2 enTNBC encompasses a heterogeneous group of breast cancers with distinct clinical characteristics, transcriptomic and genomic features, and unique histopathological differences marked by the lack of expression of ER, PR, and HER2 . TNBC reThe heterogeneous transcriptomic landscape of TNBCs was resolved in-depth in two landmark publications by Lehman and colleagues in 2011 and 2016 , 2016. BThe stratification of TNBCs into relevant molecular and clinical subtypes could prove helpful in improving treatment prediction and prognostic algorithms. Several studies analysed the response of TNBC subtypes to neoadjuvant chemotherapy and found significant differences. For instance, a retrospective study by Masuda and colleagues revealed vast differences, with basal-like type 1 TNBCs achieving pCR in 52% of cases and basal-like type 2, androgen receptor and mesenchymal TNBCs achieving pCR rates of 0, 10, and 23%, respectively . HoweverBRCA1 mutated—75% of women carrying germline mutations of this gene develop TNBCs  and with poor prognosis  was enriched in Tamoxifen-resistant cells, correlating with an increased Wnt signaling activation (Tamoxifen (active metabolite 4-OH tamoxifen [4-OHT]), a selective estrogen receptor modulator, is the first clinically approved ER-targeted agent and the most widely used hormonal treatment for breast cancer in both pre- and post-menopausal women . Despitetivation .Despite HER2 targeting agents, such as trastuzumab, substantially improved the prognosis of the HER2 subtype, the development of resistance and tumor recurrence remains a major concern. Possible mechanisms explaining resistance to trastuzumab include—but are not limited to—overexpression of other HER family receptors, increased expression and activity of c-Met, and loss of tumor suppressor phosphatase and tensis homolog (PTEN) .in vitro, an upregulation that was correlated with increased activation of Wnt signaling. In line with those findings, knockdown of Wnt3 decreased EGFR expression, rescued trastuzumab resistance, and reduced invasiveness , which have been hypothesized to significantly play a role in resistance to therapy , have been reported to have a strong correlation with the overexpression of HER2 in different breast cancer models . Korkayasiveness . Moreove in vivo .APC gene led to stabilization and accumulation of β-catenin and constitutive transcriptional activation of Wnt/β-catenin target genes through TCF/LEF:β-catenin activity   .The cause of such aberrant activity of the Wnt/β-catenin pathway in TNBC in the absence of known driver mutations is still not fully understood. However, over recent years, the pieces of this puzzle are being put together, one at a time .Some studies suggest that elevated β-catenin levels in breast cancer could be partially due to loss of Cadherin 1 (CDH1), a frequently observed feature in advanced and invasive tumors . In normLGR6, FZD6, WNT9A, WNT3A, RSPO2, SFRP1, and DKK4  promotes HMEC transformation and tumor formation through a Notch-dependent mechanism  is directly regulated by WNT/β-catenin signaling at different levels. FZD1 and Pygopus Family PHD Finger 2 (PYGO2), a recently discovered co-activator of Wnt/β-catenin -dependent transcription, were found overexpressed in chemotherapy-resistant breast cancer cell lines and to be essential for the maintenance of MDR1 expression. Interference and silencing of FZD1, PYGO2 and/or β-catenin ultimately restored sensitivity to doxorubicin , 2016.In addition to directly regulating the expression of drug-resistance mediating efflux pumps, aberrant Wnt signaling seems to be involved in controlling tumor immune suppression by regulating the exclusion of infiltrating lymphocytes from the tumor microenvironment . As mentWnt signaling has also been shown to regulate PD-L1 expression in TNBC cells, which plays a critical role in mediating tumor immune evasion . High PDOne of the central functions of Wnt/β-catenin in stem cell biology is the regulation and maintenance of self-renewal and pluripotency and the governance of differentiation fate. Wnt signaling regulates both maintenance of pluripotency and lineage specification in embryonic stem cells and early embryonic development . Small mThe involvement of Wnt signaling in the maintenance and regulation of the CSC phenotype has been intensely studied in recent years. Consequently, like in healthy stem cells and embryonic stem cells, it has been shown to regulate a myriad of functions that ultimately contribute to the survival of CSCs, which contributes to drug resistance and relapse.+ and CD24low/CD44high CSCs promoting resistance to carboplatin , which confers metastasizing properties to carcinoma cells, has also been shown to regulate the acquisition of CSC properties through Wnt regulation. Specifically, Snail has been shown to induce resistance to taxol treatment in breast cancer cells by mediating upregulation of miR-125b through the Wnt/β-catenin/TCF7L2 axis. Moreover, miR-125b overexpression was shown to expand CSC populations, and depletion of expression of this miRNA led to the reinstatement of taxanes sensitivity . RecentlThe hypoxic tumor microenvironment also regulates the CSC phenotype through Wnt signaling. Specifically, HIF-2a expression stimulated by hypoxia in breast cancer cells significantly induces paclitaxel resistance. Moreover, HIF-2a exogenous overexpression is accompanied by stem cell marker expression and enhanced Wnt activity. Importantly, inhibition of Wnt signaling by DKK1 reverts stemness and paclitaxel resistance . More reThe impact of Wnt signaling on tumour initiation, progression, and resistance to treatment is becoming evident, making it a potential therapeutic target for Wnt-driven malignancies and those where Wnt is shown to mediate CSC maintenance and/or treatment adaptation, resistance, and recurrence.Several pharmacological Wnt inhibitors have been developed to target different members or steps in the signal transduction cascade, from ligand biogenesis to transcriptional activation .Given the many functions Wnt exerts in normal tissues such as the intestine, hematopoietic system, and skin, the development of efficient and highly specific therapeutic tools is invariably hindered by the challenge of warranting minimal pharmacotoxicity and optimal tolerability. Notably, the intrinsic need of adult tissue stem cells for tight Wnt signaling regulation poses a major hurdle to the clinical feasibility of Wnt signaling inhibition due to the significant hypothetical and demonstrated side effects. One example is the use of tankyrase inhibitors and their severe gastrointestinal side effects . The samInhibitors of PORCN have been given substantial attention and tested in various cancers. Given the sheer size of the Wnt and FZD family of ligands and receptors and the high level of complexity of ligand-receptor interactions, the possibility of a “shotgun” approach that disrupts the activity of all ligands is highly desirable. This approach likely prevents compensatory upregulation of alternative receptors or agonistic ligands and the acquisition of mutations leading to alterations in antibody binding affinity. Notwithstanding, the use of such small molecules is always liable to the development of resistance through upregulation drug efflux pumps.Nonetheless, Liu and colleagues first reported the effectiveness of PORCN inhibitor LGK974 in MMTV-Wnt1 triggered murine breast cancer. Notably, their study reported no relevant intestinal toxicity at the therapeutic dose, with significant tumor reduction . This moIn addition to inhibiting their secretion, Wnt ligand activity can be disrupted by Wnt-targeted monoclonal antibodies (ipafricept—OMP54F28) and FZD-targeting monoclonal antibodies (vanctitumab—OMP18R5) . IpafricDownstream inhibitors of the Wnt signaling pathway have also been given extensive attention. A variety of molecules have been developed to target different intracellular regulators of Wnt signaling. Tankyrase inhibitors such as XAV939, IWR1, JW55, and G007-LK have shown preclinical efficacy but failed to move into clinical trials due to toxicity in preclinical studies . SeveralOne crucial limitation and challenge towards establishing Wnt signaling-targeting drugs as therapeutic options in general and breast cancer specifically is the necessity to identify and stratify in which patients the risk-benefit relationship is reasonable. To that end, the discovery and validation of robust biomarkers to predict response to Wnt-targeted treatments would prove invaluable. Interestingly, a clinical trial has reported a four-gene signature  as a predictor of response to paclitaxel/vantictumab combinatorial treatment in HER2 negative breast cancer . A testaFrom the earliest mammary morphogenic events to drug-resistant breast cancer, our knowledge of the importance of Wnt signaling in the breast keeps increasing. The virtual inexistence of well-characterized Wnt-driving genetic mutation casts a shadow over the functional relevance of aberrations in Wnt signaling regulation in breast cancer. Discovering the provenance of such aberrant levels of Wnt signaling activation could prove invaluable in the ongoing efforts to discover druggable targets for TNBC.Exploiting the relationship between aberrant Wnt activity and enhanced cancer stem cell function and drug resistance has long been regarded as a potential therapeutic avenue for this breast cancer subtype. However, our current understanding of the underlying pathobiology of aberrant Wnt signaling is shallow since so much is yet to be understood about the regulation of this intricate signaling cascade in healthy conditions. Notwithstanding, understanding whether aberrant Wnt signaling is an intrinsic feature of breast cancer, perhaps harbored by underrepresented cancer cell populations, or a feature of populational evolution and treatment adaptation by non-mutational mechanisms is an important question that needs to be addressed to untap the potential for Wnt signaling targeting therapies in breast cancer. Relevant preclinical research models will be critical towards resolving the underlying Wnt signaling heterogeneity of breast cancers. Murine models of breast cancer differ from human breast neoplasms in many ways, including their immune environment, vascularization, and tumor microenvironment, among others. All of these can contribute to the disconnects often observed in the translation of fundamental research studies into the clinic .Due to the myriad of Wnt-dependent physiologic functions, pharmacological modulation of this pathway remains particularly challenging. As such, mapping the functional outcomes of Wnt signaling perturbations in breast cancer cell populations to dissect possible points of pharmacological intervention is necessary to circumvent organism-wide side effects of Wnt signaling inhibition.Understanding which patients are more likely to benefit from Wnt targeted therapies is just as important has gaining deeper knowledge of the underlying biology of the Wnt signaling pathway. The selection of such patients for Wnt targeted therapies requires a serious commitment toward the development of stratification protocols based on robust biomarkers and detection assays.Wnt signaling-targeting therapies could undoubtedly carve out an important place in the breast cancer therapeutic arsenal. However, given the current pharmacological state-of-the-art and the overall complexity of Wnt signaling activation in normal and disease conditions, significant research is needed to enable Wnt-targeting drugs as suitable therapeutic tools.


Human T-lymphotropic viruses 1 and 2 (HTLV-1 and HTLV-2) are retroviruses that originated on the African continent and dispersed throughout other continents through human migratory flows. This study describes the prevalence of HTLV-1 and HTLV-2 infection in residents of 11 quilombo remnant communities in the state of Pará, Brazil, and the associated risk factors. A total of 859 individuals (334 men and 525 women), aged between 7 and 91 years, participated in the study. All subjects answered a questionnaire with questions on sociodemographic characteristics and on risk factors associated with HTLV infection, and blood samples were collected and separated into plasma and leukocytes. An immunoenzymatic assay  was used as a screening test, and positive samples were subjected to line immunoassay confirmatory tests (Inno-LIA HTLV I/II Score FUJIREBIO) and DNA extraction for subsequent real-time PCR to differentiate the viral type. Four of the 859 individuals were seropositive for HTLV. HTLV-1 infection was confirmed in one individual from the Itamoari community (0.92%), and HTLV-2 infection was confirmed in two individuals from São Benedito (3.17%) and in one individual from Arimandeua (2.22%). Blood transfusion was the only risk factor associated with HTLV infection in this study. This study reports the occurrence of HTLV-1 and HTLV-2 in quilombo remnant communities in the state of Pará. Considering the African origin of the virus and its introduction into Brazil from the slave trade, the continued evaluation of quilombola communities in the state of Pará is essential to better characterize the distribution of infections in these populations and to create public health policies for the control of the spread of the virus and associated diseases. Retroviridae, subfamily Orthoretrovirinae, and genus Deltaretrovirus (Human T-lymphotropic viruses 1 (HTLV-1) and 2 (HTLV-2) were the first isolated retroviruses to infect humans , Umarizal Beira (n = 303), Arimandeua (n = 45), Aripijó (n = 31), Bacuri (n = 10), Cabanagem (n = 17), São Benedito (n = 63), Bela Aurora (n = 35), Camiranga (n = 89), Itamoari (n = 109), and Nova Jutaí (n = 137)  were visited from September 2020 to April 2021, and information was collected from 859 individuals. The following communities were studied: Poeirinha (n = 137) .All study participants signed an informed consent form and answered a questionnaire with questions about sociodemographic aspects  and behavioral risk factors for exposure to HTLV . Individuals under 18 years old signed an informed assent form, and their legal guardians signed an informed consent form.From all participants, peripheral blood samples (4 mL) were collected in vacuum tubes containing ethylenediaminetetraacetic acid (EDTA) as an anticoagulant. The samples were then subjected to centrifugation to separate the plasma and leukocyte fractions, followed by storage at −20°C until use.This study was approved by the National Research Ethics Committee  (CAAE: 27290619.2.0000.0018), in compliance with resolution 466/12 of the Ministry of Health, which regulates research involving human beings. All subjects who voluntarily agreed to participate in the study signed an informed consent form.® HTLV I/II Score, Fujirebio, Japan) and real-time PCR  to confirm infection and differentiate the viral type, respectively.Immunoenzymatic assays  were used to detect anti-HTLV-1/2 antibodies in the tested samples following the manufacturer's instructions. Samples with positive results were subjected to line immunoassays  following the manufacturer's protocol. Molecular confirmation of infection was performed using a multiplex real-time PCR for three target sequences: the albumin gene (141 bp), as an endogenous control; the pol gene (186 bp) of HTLV-1; and the tax-2 gene (75 bp) of HTLV-2. The primer sequences used were 5′-ccctacaatccaaccagctcag-3′ (HTLV-1F), 5′-gtggtgaagctgccatcgggtttt-3′ (HTLV-1R), 5′-cgattgtgtacaggccgattg-3′ (HTLV-2F), 5′-caggagggcatgtcgatgtag-3′ (HTLV-2R), 5′-gctgtcatctcttgtgggctgt-3′ (F Albumin), 5′-aaactcatgggagctgctggtt-3′ (R Albumin); the probe sequences used were JOE-5′-ctttactgacaaacccgacctacccatgga-3′-BHQ (HTLV-1), FAM-5′-tgtcccgtctcaggtggtctatgttcca-3′-MGB (HTLV-2), and NED-5′-cctgtcatgcccacacaaatctc-3′-MGB . The reaction components, based on the TaqMan® Universal system, were as follows: 12.5 μL of Master Mix, 6.0 μL of water, 1.0 μL of Assay-by-Design (primer and probe set) and 0.5 μL of DNA in a final volume of 20 μL. For cycling, the protocol was as follows: 50°C for 2 min; 95°C for 10 min; and 50 cycles of 90°C for 50 s and 60°C for 1 min.To perform real-time PCR, DNA was extracted from the seropositive samples using a The collected information was stored in a database using Epi-Info7.2 software. The socioeconomic aspects, behavioral factors, and risk factors for exposure to HTLV-1 and HTLV-2 are described as frequencies and percentages. The confidence interval (95% CI) was used to estimate overall and community prevalence. Fisher's exact test was used to evaluate the association of behavioral risk factors with the presence/absence of HTLV-1 or HTLV-2. The significance level adopted was 5%. Statistical analyses were performed using the programs BioEstat 5.3, GraphPad Prism 7.0 for Windows, and MINITAB Release 14 for Windows.n = 525) of the individuals were women, and 38.9% (n = 334) were men. A total of 49.9% (n = 429) of the individuals said they were married/living with a partner. Most of these individuals declared themselves black  and brown . Regarding education, 40.6% (n = 349) were literate, 16.5% (142) had completed elementary school, and 28.4% (n = 244) had completed secondary education. A total of 46.6% (n = 400) live on <1 (1) minimum wage, and 47.1% (n = 405) live on one (2) to three (3) minimum wages ; 61.1% . p = 0.0176) and HTLV-1/2 infection, with no association with any of the other factors investigated . Three individuals (75%) denied having used illicit drugs, and one did not report anything. All of them denied having piercings, a previous diagnosis of an STI, or having more than one sexual partner. Two (50%) of those infected reported having received blood transfusions. Half 5%. Three. Accordistigated .Herein, we report the occurrence of anti-HTLV-1/2 antibodies in four of the 859 individuals tested, with confirmed HTLV-1 infection in only one resident of the Itamoari community and confirmed HTLV-2 infection in two communities—Arimandeua and São Benedito. This is the first time that HTLV-1 and HTLV-2 have been identified in these communities, and no previous studies have been conducted in these communities.Vallinoto et al.  found thThe prevalence of HTLV-2 in the present study was lower than that reported by other studies involving traditional populations of Pará, such as indigenous populations. The prevalence in three Kayapó villages in the Xikrin territory was 29%, ranging from 21 to 38% among the villages . In turnItamoari is a community located on the Pará side of the Gurupi River. The Gurupi River and the Turiaçu River (State of Maranhão) form a region called Turiaçu. The port in this region served as an entrance for a large number of slave through illegal slave trade during the 19th century . In thisThere are historical reports  that slaHistory of an STI, multiple sexual partners, and non-use of condoms are risk factors that have been associated with a higher risk of HTLV-1/2 infection; however, in the present study, no associations were found , 25, 53.Blood transfusion was a risk factor for HTLV-1/2 infection in this study as well as in other studies , 55. In All three HTLV-2-infected patients have one surname in common; thus, it is possible that there is some degree of kinship between them due to the isolation of these communities. In addition, the 67-year-old woman who was infected reported that she had 11 pregnancies and breastfed all of her children for more than 6 months. However, her children could not be found to be tested, and thus, it is not known whether there was vertical transmission of the virus or if her infection occurred after her pregnancies. A new visit to the community is being arranged to investigate intrafamily transmission.Although most of those infected with HTLV-1 are asymptomatic, this virus is associated with clinical manifestations that can lead to severe symptoms and death, and there is no cure or treatment available for the infection, which is neglected, not only in Brazil but also in several regions of the world . TherefoAlthough many Brazilian population segments are in a situation of vulnerability, there is a higher prevalence of black populations among the vulnerable populations, including those living in quilombo remnant communities , 58. TheConsidering the occurrence of HTLV-1/2 infection in populations associated with the African origin of the virus and its introduction into Brazil from the slave trade, the continuous evaluation of quilombola communities in the state of Pará and other locations in the Brazilian Amazon is needed to better characterize the distribution  of infection in these populations to better formulate public health policies to control the spread of the virus and associated diseases.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Human Research Ethics Committee of the Health Sciences Institute of the Federal University of Pará (CAAE: 55699316.6.0000.0018). The patients/participants provided their written informed consent to participate in this study.AV, JG, ES, IC, and GC-C conceived and designed the study. WB, LR, KP, FL, IA, CL, and AL assisted in subject recruitment and sample collection. BS, FL, WB, IA, and CL performed the serological and molecular analyses. SL and WB performed statistical analyses. WB, GC-C, IC, and AV wrote the manuscript. All authors read and approved the final manuscript.This study was supported by the National Council for Scientific and Technological Development  and the Federal University of Pará(PAPQ-2022).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Moreover, the Δatg1Δpmk1double mutant resembles the single Δpmk1 mutant, suggesting that Atg1 functions in the Pmk1 pathway. In addition, the growth defect induced by overexpression of Pck2, an upstream activator of Pmk1 MAPK was alleviated by the deletion of+atg1. Finally, the deletion of autophagy-related genes recapitulates Pmk1 MAPK signaling inhibition. Our data suggest a novel role for autophagy in MAPK signaling regulation.Apart from the highly conserved role in the cellular degradation process, autophagy also appears to play a key role in cellular proliferation. Here, we describe the genetic interaction of autophagy-related genes and Pmk1 MAPK signaling in fission yeast. Although starvation potently induces autophagy, the basal level of autophagy is maintained even in normal growth conditions, thereby controlling development, cellular metabolism, and proliferation in all eucaryote cells . However, how autophagy organizes cellular signaling networks to exert these functions is not fully understood.Autophagy is an evolutionarily highly conserved mechanism to maintain cellular homeostasis via waste clearance using the lysosomal machinery  phenotype, which recapitulates Pmk1 MAPK signaling inhibition (Methods). The rationale of thevicmutant screening is based on the antagonistic relationship between Pmk1 MAPK and calcineurin, a highly conserved serine/threonine protein phosphatase, in the Cl−homeostasis in fission yeast. Knockout of theS. pombecalcineurin geneppb1+ or inhibition of calcineurin activity by the immunosuppressant FK506, a specific inhibitor of calcineurin, results in hypersensitivity to Cl−. This phenotype associated withppb1deletion was reversed by the inhibition or gene knockout of the components of the Pmk1 MAPK signaling pathways, including Pmk1 MAPK, Pek1 MAPKK, Mkh1 MAPKKK, and Pck2 Protein kinase C . Our previous genetic screening to isolatevic mutants identified upstream activating regulators of MAPK signaling such as geranylgeranyl transferase  and farnesyl transferase .Here, we identified2, whereas knockout of thepmk1+gene makes cells grow much faster in the same condition . Δatg1 cells grew faster than the WT cells in the presence of FK506 and 0.09 M MgCl2, although the growth of Δatg1 cells was slower than that of Δpmk1. The WT, Δpmk1 and Δatg1 cells exhibited essentially the same pattern of sensitivity and resistance to FK506 and Cl−on EMM, YPD and YES plates, although the sensitivity of the WT cells and Δatg1 cells was more enhanced on YES than the other plates. Genetic interaction between Δatg1and Δpmk1was further examined by constructing Δatg1Δpmk1double mutant cells. The degree of thevic phenotype in Δatg1Δpmk1double mutants and Δpmk1cells was almost equivalent. These results are consistent with Atg1 working upstream of the Pmk1 pathway. The difference in growth of these strains is almost indiscernible in the absence of FK506 and MgCl2.The growth of the wild-type (WT) cells was significantly inhibited in the presence of the calcineurin inhibitor FK506 and 0.09 M MgClet al.2006). This phenotypic evaluation also led to the identification of an SH3 adaptor protein Skb5 as a negative regulator of Pck2/Pmk1 signaling . As shown in Figure 1B,atg1deletion significantly suppressed the toxicity induced by Pck2 overexpression, although the impact ofatg1deletion on the suppression of the toxicity of Pck2 overexpression was smaller than that achieved bypmk1deletion. Thus,atg1 deletion is likely to ameliorate Pck2-mediated Pmk1 MAPK hyperactivation.To further explore the functional relationship between Atg1 and Pmk1 MAPK signaling, we utilized the cell growth inhibition associated with Pck2 overexpression. Pck2 overexpression in the WT cells leads to Pmk1 MAPK hyperactivation and cytotoxicity, which can be suppressed by the inhibition or knockout of the components of the Pmk1 MAPK pathway  . The degree of thevic phenotype of theseatgmutants was similar to that of Δatg1 cells. These results suggest that the autophagy system as a whole may be involved in the Pmk1 MAPK signaling regulation.Next, we confirm if the loss-of-function mutants of other autophagy-related genes also display theet al.2006), as well as the role of autophagosome as a scaffold to facilitate spatial coordination of RAF/MEK/ERK phosphorylation . Our epistasis analysis, showing the nonadditivevicphenotype of Δatg1Δpmk1double mutant and the resemblance of the Δatg1Δpmk1double mutant to the Δpmk1single mutant, suggests that Atg1 acts upstream of the Pmk1 signaling pathway. Furthermore, suppressioin of the toxicity induced by Pck2 overexpression byatg1deletion suggests that Atg1 acts downstream of Pck2. MAPK signaling cascades consist of a core module of three tiers of protein kinases MAPK, MAPKK, and MAP3K, and often an additional upstream MAP4K. It would be intriguing if Atg1 serves as an additional layer of kinase mediating MAPK signaling activation. Future studies will elucidate the mechanism and the functional significance of the genetic interaction between autophagy and MAPK signaling revealed by our yeast genetic screen. Given the highly conserved nature of autophagy and MAPK signaling in the fate of cell death and proliferation, this study will provide valuable information to understand human diseases associated with aberrant regulation of MAPK signaling and autophagy.In summary, our genetic screen revealed a functional interaction between autophagy-related genes and Pmk1 MAPK signaling in fission yeast. Several studies report functional crosstalk between MAPK signaling and autophagy, including the role of MAPK ERK in the maturation of autophagosomes . FK506 was provided by Fujisawa Pharmaceutical Co. . Standard genetic and recombinant-DNA methods  were used except where noted.vicmutant screeninget al. was analyzed on the YPD plates with or without FK506 and 0.09 M MgCl2by spotting growth assay.The growth of 89 viable kinase knockout strains generated by BimboSpotting growth assay660/ml, from which five 10-fold serial dilutions were prepared and then spotted onto the indicated plates. Plates were incubated at 27˚C for 3 to 7 days.Yeast cells were cultured in 20 ml of liquid media at 27˚C till mid-log phase and diluted to 0.6 OD


We optimized the vesicle stabilityand antibacterial activity by tuning the surfactant chain length andheadgroup charge (cationic or zwitterionic) and show that these quatsomescan furthermore serve as nanocarriers of pharmaceutical actives, demonstratedhere by the encapsulation of (+)-usnic acid, a natural substance withmany pharmacological properties.The efficacy of thetreatment of bacterial infection is seriouslyreduced because of antibiotic resistance; thus, therapeutic solutionsagainst drug-resistant microbes are necessary. Nanoparticle-basedsolutions are particularly promising for meeting this challenge becausethey can offer intrinsic antimicrobial activity and sustained drugrelease at the target site. Herein, we present a newly developed nanovesiclesystem of the quatsome family, composed of  Quatsomes have great potential in biomedical applicationsbecause they share some important features with liposomes but overcomesome problems such as a lack of stability and aggregation over time.In fact, quatsomes are very stable and homogeneous in lamellarityand size, features that do not change in a wide range of temperaturesor dilutions.N-oxide surfactants, with alkyl chain lengthsof 12, 14, or 16 methylenes, respectively . These surfactants are l-prolinol-derived surfactants, which were shown to enhance theefficacy of DNA and other lipid drug-delivery systems,12 reduce the effective dose of some antibiotics13 and confer antibacterial activity to liposomal formulations.14 The possibility of including these surfactantsat high molar percentage in the formulations can maximize their pharmacologicalpotential. We exploited depressurization of an expanded liquid organicsolution–suspension (DELOS-SUSP), a green and scalable methodologythat allows one to work in sterile conditions.3 With this one-step technique, it is possible to obtain unilamellarnanoscale quatsomes showing narrow size distribution and more homogeneouscomposition compared to those obtained with conventional vesiclespreparation protocols.16 It consists of depressurizationof a CO2-expanded solution containing chol, dissolved inacetone or ethanol, into an aqueous phase containing the quaternaryammonium surfactant. The use of supercritical fluids grants easiercontrol of the morphology and the dimensions of the nanovesicles inorder to achieve high reproducibility.1 (+)-Usnic acid  substance producedby many lichens, was also loaded into the formulations. Unfortunately,UA is scarcely soluble in water21 and showsdose-dependent hepatotoxicity.22 As a consequence,its application has been limited to topical ointments, oral care productsor cosmetic formulations.25 It is clear that the inclusionof UA in drug-delivery systems would take advantage of its severalpharmacological properties upon systemic administration. For thesereasons, we evaluated the entrapment efficiency (E.E.) of UA and itsantibacterial activity when included in quatsomes. In particular,the antibacterial effectiveness of these nanovesicles, including ornot UA, was evaluated on Gram-positive and Gram-negative bacteriaand fungal strains. In a previous investigation, liposomes containinganalogous structurally related l-prolinolderivatives showed a good ability to efficiently deliver UA to S. aureus bacterial cells.10 Becausethe biological activity of UA in some cases can be related to theantioxidant one,26 we assessed also theantioxidant effectiveness of quatsomes/UA. In fact, we previouslyshowed that this property can significantly vary as a function ofthe composition of the systems in which it is included.31 The same evaluation was carried out in liposomes prepared eitherwith thin-film hydration (TFH)32 or withDELOS-SUSP33 and formulated with 1,2-dimyristoyl-sn-glycero-3-phosphocholine, chol, and 10 mol % of the samesynthetic surfactants as those used in this investigation. The aimof this study was to correlate the physicochemical properties of theaggregates with their biological effects to individuate which factorsmostly affect the ability of the formulations to interact with thebiological milieu and to optimize their potentialin terms of drug-delivery efficacy.Here we report on the preparation of quatsomescontaining equimolaramounts of chol and one of the synthetic surfactants reported in acid UA; 1, a pharQuatsomes were prepared using DELOS-SUSPequipment. The size and ζ potential of quatsomes were measuredby dynamic light scattering (DLS) using the apparatus described inref . The wat2; 99.9% purity)was purchased from Carburos Metálicos S.A. .Sabouraud dextrose broth, Sabouraud dextrose agar (Liofilchem), andMueller–Hinton broth (Biolife) were the media used for biologicaltests. UA, RPMI-1640 with l-glutamine, phenol red powderdissolved in 3-(N-morpholino)propanesulfonic acidat a final concentration of 0.165 M and pH 7.0 supplemented with 2%glucose (RPMI-1640 G2), dialysis tubing cellulose membrane (cutoff= 14000), CH3COONa, H2O2, and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonicacid) diammonium salt (ABTS) were purchased from Sigma-Aldrich . Cationic surfactants  and their corresponding N-oxides  were prepared as reported in ref (Chol (purity 95%) waspurchased from Panreac. Milli-Q water , ethanol ,and dimethyl sulfoxide  in highpurity were the solvents used for the preparation of the samples byDELOS-SUSP equipment. Carbon dioxide (COd in ref . All solS. aureus reference strainfrom the American Type Culture Collection (ATCC 43300), Escherichiacoli (ATCC 25922), and Candida albicans (ATCC64124) fungal strain were used as control organisms.Methicillin-resistant 8 Chol (4.87 mg)was dissolved in 1.2 mL of EtOH under the conditions described inref (Tw) Milli-Q water containing around 5 mg of one of theCSs or N-oxs [molar ratio chol/CS (N-ox) = 1:1; total concentrationof membrane components = 1 mM). During depressurization, N2 (11.5 MPa) was used to flush the reactor to achieve a constant Pw. Quatsome suspensions were purified througha dialysis process to eliminate EtOH by exchanging the external medium,which was Milli-Q water at 25-fold the quatsome dispersion volume,four times within 1 h.The investigatedquatsomes were prepared by DELOS-SUSP, a methodology based on theuse of compressed fluids.d inref . The orgTw. Theproper amount of UA to obtain a final UA concentration equal to 5× 10–5 M in DMSO (50 μL) was preheatedat Tw and adjoined dropwise to the organicphase, which was then added to the high-pressure vessel. The molarratio UA/quatsome components  is 1:20. The remaining part of the protocol was the same asthat described above (without UA). Quatsomes were treated with dialysisto eliminate DMSO and unentrapped UA, besides EtOH.UA loadingin the DELOS-SUSP process: chol (4.87 mg) was dissolved in 1.15 mLof EtOH at working temperature i.e., lipid+ chol)/UA of 20:1; then the dispersion was warmed for 1 h at 40 °C.Quatsomes were treated with dialysis to eliminate the organic solvents(DMSO and EtOH) and UA not included in the aggregates.UA loadingby incubation: a few microliters of a solution of 37.5 mM UA dissolvedin DMSO was adjoined to previously prepared quatsomes to obtain afinal molar ratio  as described previously.The quatsomes morphology wasstudied by cryo-TEM measurements as described in ref .The solution containingthe radical cation was prepared as reportedin ref .•+ solution was rapidly added to 2450 μLof acetate buffer at pH 5.5 prepared as described above containing1.38 × 10–6 M of free or quatsome-loaded UA.Variation of the maximal absorbance at 417 nm was followed for 1 h.The reported results are the average of at least three repeated measurements.The absorbance decay over time was fitted using Origin Pro 2012 toquantify the contribution of UA to the degradation of ABTS•+, as described in refs (ABTS = 34.4 min).A volume of 25 μLof ABTS in refs  and 33)•+ solutiS. aureus and E. coli strains to quatsomeswith or without UA was evaluated following the CLSI guidelines.35 Briefly, for the antibacterial susceptibility,100 μL of a bacterial suspension in a 0.9% saline solution (NaCl)at a concentration of 5 × 10–5 CFU/mL was addedto the wells of a 96-well microtiter plate containing 100 μLof 2-fold serially diluted free UA and quatsomes loaded with UA (withboth loading methodologies) in cation-adjusted Mueller–Hintonbroth. The experiments were then carried out as reported in ref .36 Briefly, a glycerol stock solutionof yeast was inoculated into Sabouraud dextrose broth and subsequentlysubcultured onto Sabouraud dextrose agar. The inoculum at 0.5 McFarlandin 0.9% saline solution NaCl was prepared by picking some coloniesand following the method described in the EUCAST guidelines in orderto reach the final concentration of (2.5–0.5) × 105 CFU/mL. A total of 100 μL of inoculum was added tothe wells of a 96-well flat bottom microtiter plate containing 100μL of 2-fold serially diluted free UA and quatsomes loaded withUA in RPMI-1640 G2. The antifungal activity was determined by a microdilutionmethod in 96-well microplates statically incubated for 24 h at 35°C. Microorganism growth was spectrophotometrically quantifiedat 595 nm by a microplate reader. The minimum inhibitory concentration(MIC) for free UA and quatsomes loaded with UA was defined as theconcentration of drug that decreases growth by 80% compared with thatof organisms grown in the absence of drug. The MICs of quatsomes wereindicated through nominal surfactant concentrations that were usedfor quatsome production.The antimycoticeffect of quatsomes with or without UA against Table S1) and over time up to 4 months (Table S2). The formulations differ in their surfactant chain lengths  and/or headgroup charge . In the first weeks after preparation, all of the investigatedformulations, except chol/N-ox 12, yielded monomodaldistributions of sizes with an average hydrodynamic diameter of 50–100nm and low dispersities ,obtained from cumulant analysis (Table S1). Only chol/N-ox 12 yielded dimensions in the micrometerrange with increased dispersity. After 4 months, the dispersity remainedlow, and average diameters increased by about 30–40 nm (Table S2), with the exception of chol/CS 16, which was stable in size for 4 months and showed a sphericalunilamellar morphology after this time . Chol/N-ox 12 featured a positive potential around 50 mV soon after thepreparation (similarly to CS 12 containing quatsomes)that turned negative after 1 week, indicating a loss of stabilityof the bilayer and thus confirming the low stability of this formulation.Reasonably, in the lipid rearrangement, there is a variation of theexposure of the polar headgroup that brings a higher association ofcounterions to the positively charged group of the N-oxide moiety with consequent reduction of the ζ potentialof the formulations. Positive ζ potentials were observed forliposomes containing these zwitterionic N-ox surfactants, with theexception of N-ox 12, independently of the methodologyused for their preparation.34 Our data point outthat, when quatsomes contain CS 12, the nature of thepolar headgroup is more relevant than that for longer alkyl chains,probably because the extent of hydrophobic and van der Waals interactionsis significantly reduced. In fact, even if the alkyl chain containsonly two or four methylenes less, the aggregation number of quatsomesis very high and the overall effect is reflected not only on theirstability but also on the hydrophobic/hydrophilic balance of the aggregates.In particular, N-ox 12 forms the least stable quatsomes,indicating that the repulsive interactions among the zwitterionicsurfactants, despite their overall electrical neutrality, are higherthan those among the corresponding cationic CS 12. Literaturereports indicate that surfactants containing the N-oxide moiety and a C12 chain can destabilize the bilayer even atlow molar percentage.38 Also liposomes prepared withDELOS-SUSP containing CS 12 and N-ox 12 wereless stable than the others, even if their destabilizing effect wasless evident,34 likely because of the lowermolar fraction of the surfactant in the total composition (10 mol% in liposomes vs 50 mol % in quatsomes). Liposomeswith the same composition prepared according to TFH were considerablyless stable,33 even more than quatsomesthat contain a 5-fold amount of synthetic surfactant. Even if N-ox 12 and the other surfactants with longer alkyl chains differby only two or four methylenes, it is not so unusual that this differencecan be crucial in determining properties of the aggregates that formor in which they are included,38 Also micelles formed by the samesynthetic surfactants used in this investigation show different propertiesas a function of the chain length.27 Moreover,many literature reports on aggregates containing or formulated withsurfactants with unrelated molecular structure from the investigatedones but differing for only two or four methylenes in the hydrophobicchain demonstrate that this parameter can play a pivotal role in determiningthe properties of the aggregates.43 As a whole, the obtained results confirm the following:(i) the high stability of quatsomes containing surfactants bearingat least 14 carbon atoms in the chain (even in this case in whichcationic or zwitterionic synthetic surfactants were used for the firsttime for their preparation); (ii) the high stability and homogeneityof the bilayer of the aggregates prepared using DELOS methodology;(iii) the pivotal role of the hydrophobic/hydrophilic balance in determiningthe nanovesicle properties.All formulations exceptchol/N-ox Table S1 and also Table S3). The dialysis (carried out 1 week aftertheir preparation) did not affect the dimensions of the nanovesiclesexcept for CS 12 quatsomes, which showed, like the N-ox 12 ones, an increase of the dimensions and PDI value, togetherwith incipient precipitation (data not shown). Different from liposomes,33 for quatsomes the high amount of synthetic surfactant (50 mol %)seems to mask the effect of UA on the ζ potential. Moreover,the absence of the phospholipid (that bears two long chains) couldmake the bilayer slacker with respect to liposomes, allowing UA tomore deeply penetrate the hydrophobic region. In fact, it is well-knownthat the contribution to membrane mechanics in a bilayer is strictlydependent on the chemical structure of the lipid/lipids that composeit.44 All of the samples mainly showeda slight decrease (about 10 mV) of ζ potential over time, moreprominent (30 mV) in the case of CS 12 quatsomes probablybecause of their lower stability which leads to lipid rearrangement.A similar decrease of the potential for the sample containing CS 12 was observed upon dialysis 1 week after the preparation,thus confirming the low stability of the formulation.The inclusion of UA in the formulations barely affectedtheir sizeor ζ potential, regardless of whether UA was added during vesicleformation or by incubation (Table S4), indicating that UA can penetrate the bilayer and its presencedoes not disturb lipid organization. The only exception was the formulationcontaining CS 12, in which UA was included in the vesselduring formation of the vesicles: in this case, the E.E. was around50%. In general, this formulation showed a different behavior overtime and when subjected to dialysis with respect to the samples containingCSs with longer chains. Moreover, the high E.E. observed through incubationof UA suggests a different location of this molecule in the bilayeror a different permeability depending on the methodology of the preparation.All ofthe formulations showed very high E.E. (12 showed a high E.E. (both by the addition of UA in the reactorand by incubation) even if the ζ potential in the last casewas negative. It is possible that the low stability of this sampleimplies a reduction of lipid packing and thus UA can more easily penetratethe bilayer.An interesting result is that the formulation containing N-ox 49 so we decided to assess the antioxidant activity of UA includedin quatsomes according to a commonly used assay.34 Briefly, the radical cation of ABTS .The antioxidant activityof natural compounds can significantly vary when they are embeddedin a lipid bilayer,expected 2A,B, whe•+ in the presence of N-ox 12 probablybecause of a different organization and/or compaction of the bilayer.For the samples containing surfactants bearing a C16 chain, the trendwas respected but complex phenomena occurred, especially if UA wasincubated. The chain length and nature of the polar headgroup significantlyinfluence the antioxidant activity of UA also when it is includedin pure micelles formed by the same surfactants used in this study.27 In general, the polarity and rigidity of themicroenvironment experienced by a compound play a crucial role inits exerted antioxidant activity.52 The same molecules inspherical or rodlike micelles of the same composition show differentantioxidant activities as a function of their different locationsinside the aggregates.53 The results thatwe obtained with UA can be due to the different ζ potentialsof quatsomes and/or to the different locations of UA inside the aggregates.To better understand these aspects, we also fitted all of the curveswith a double-exponential decay, considering the spontaneous and UA-relateddegradation of free ABTS•+. The time constant τUA of the UA-related degradation of free ABTS•+, reported in 32 UA contributes about 30–40%to the process  reduces the hydrophobic andvan der Waals interaction among lipids thus making the bilayer morepermeable.In general, formulations containing N-oxs showedhigher antioxidantactivity than those containing CSs. The activity was stronger forN-oxs with shorter chain lengths (12 and 14). Similar effects wereobserved independently of the preparation method of the UA-loadedquatsome formulations 2A,B. How process 2D, espec12) was evaluated on methicillin-resistant S. aureus  and E. coli  bacterial strains  and on a C. albicans fungal strain. On C. albicans (ATCC 64124). We did not observe any effectfor any of the investigated formulations except for chol/CS 16, which showed a small growth reduction at a total lipidconcentration of about 1 × 10–3 M (0.5 ×10–3 M of surfactant). On S. aureus, quatsome formulations containing surfactants bearing C14 or C16chains showed a MIC of about 2.5 × 10–5 M ,whereas samples containing CS 12 showed a MIC of about1 × 10–4 M (0.5 × 10–4 M of surfactant). On E. coli, in the presence ofsurfactants CS 14, CS 16 and N-ox 14, the observed MIC was about 1 × 10–4 M (0.5 × 10–4 M of surfactant), whereas forthe formulations chol/CS 12 and chol/N-ox 16, it was about 2.5 × 10–4 M (1.25 × 10–4 M of surfactant). In general, the antibacterial activityof the formulations increases with the ζ potential, which isknown to promote interaction of the aggregates with bacterial membranes.The antibacterial activity of the differentquatsome formulations , for S. aureus, the MICs cover the range from 8.7 μg/mL(2.5 × 10–5 M chol/N-ox 14) to37.5 μg/mL (1 × 10–4 M chol/N-ox 12) whereas for E. coli, the MICs are inthe range of 35 μg/mL (1 × 10–4 M chol/N-ox 14) to 93.8 μg/mL (2.5 × 10–4 M chol/CS 12). Consequently, these formulations, althoughnot antimicrobial molecules but supramolecular aggregates, have MICvalues that are comparable to those of several antibiotics used inclinical practice.35In all cases, the presence of UA in the formulationsdid not alterthe MIC values independently from the methodology used for UA loading.The low UA concentrations at the MICs of these quatsomes  make itThe resultsobtained by the biological experiments are encouragingbecause they highlight the relevant potential of the investigatedformulations for antibacterial treatments. Further detailed investigationson the cytotoxicity of the formulations toward mammalian cells andspecific studies to ascertain the mechanism of interaction will becarried out on the most promising formulations.l-prolinol-derived surfactants were prepared and characterized.As a whole, the supramolecular nanometric structures containing longerchains (C14 and C16) were very stable and showed a good ability toentrap UA, demonstrating high potential as a drug-delivery system.UA included in quatsomes maintained its antioxidant activity, whichwas particularly strong for N-ox formulations bearing short chainlengths. Moreover, the investigated quatsomes showed good antibacterialactivity even on Gram-negative bacteria, demonstrating that they aregood candidates for the treatment of bacterial infections alone orin association with an active principle that they can load.Nanoscale quatsomes formulated with structurally related


The Hippo signaling pathway is involved in cell growth, proliferation, and apoptosis, and it plays a key role in regulating organ size, tissue regeneration, and tumor development. The Hippo signaling pathway also participates in the occurrence and development of various human diseases. Recently, many studies have shown that the Hippo pathway is closely related to renal diseases, including renal cancer, cystic kidney disease, diabetic nephropathy, and renal fibrosis, and it promotes the transformation of acute kidney disease to chronic kidney disease (CKD). The present paper summarizes and analyzes the research status of the Hippo signaling pathway in different kidney diseases, and it also summarizes the expression of Hippo signaling pathway components in pathological tissues of kidney diseases. In addition, the present paper discusses the positive therapeutic significance of traditional Chinese medicine (TCM) in regulating the Hippo signaling pathway for treating kidney diseases. This article introduces new targets and ideas for drug development, clinical diagnosis, and treatment of kidney diseases. Drosophila melanogaster, the Hippo pathway was first identified 20 years ago during tissue growth screening  consists of clear cell renal cell carcinoma (ccRCC), accounting for 70%–80% of kidney cancers . Sheets,ogenesis . Other rogenesis . The Hipogenesis . Studiesosophila . In highosophila . Mucinouosophila . Moreoveosophila , and inhosophila . In vitrpression . By inhipression . Claudinpression . Claudinpression . Quakingpression . Immunohpathways . Moreovepathways . KIBRA, pathways . The relpathways .−/− cell transcriptome and pre-cystic kidney transcriptome indicate the involvement of the Hippo signaling pathway in early polycystic kidney disease. In vitro and in vivo Pkd1del models show changes in MAPK, Wnt, Hippo, PI3K/Akt, and calcium signaling pathways related to ADPKD  and autosomal recessive polycystic kidney disease (ARPKD), which are cilia-related diseases . ADPKD ito ADPKD . At the to ADPKD , and delto ADPKD . NIMA-reto ADPKD . Treatmeto ADPKD . NEKs reto ADPKD . Sortingto ADPKD . A membeto ADPKD . The TAZto ADPKD . TAZ indto ADPKD . In Pkd1to ADPKD . RhoA/YAto ADPKD . Severalto ADPKD . A new sto ADPKD .Worldwide, AKI has a high mortality rate (over 50%)  with an in vivo regulation of connection and migration processes is the basic mechanism of glomerulosclerosis and renal function loss. Rac1, a member of the Rho family of GTPases, plays an important role in cell movement, cell bone remodeling, and cell cycle transport . Approximately 30% of patients with type 1 diabetes and 20%–50% of patients with type 2 diabetes may develop DKD . Thus, st of DKD . Many ext of DKD . Accordit of DKD . The prot of DKD . High glt of DKD . YAP actt of DKD . In addit of DKD . The exit of DKD  , the Hippo-Salvador signaling pathway increases renal tubule interstitial fibrosis . In addiThe Hippo signaling pathway regulates cell growth, proliferation, and apoptosis as well as organ, tissue, and tumor development . AnalysiTriptolide (TP) is a diterpenoid trioxide compound that has anti-inflammatory and immunomodulatory properties . TP is cQuercetin is a type of biological flavonoid compound, and it has a variety of pharmacological effects, including reduction of the risk of kidney and cardiovascular diseases as well as antitumor, antioxidant, antiviral, and anti-inflammatory effects . In receAlthough TP and quercetin have contradictory functions in many biological processes, they both regulate kidney disease by modulating the Hippo signaling pathway. Through its ability to regulate the Hippo signaling pathway, TP reduces renal tubular epithelial fibrosis . The HipNephropathy research has shown that some nephropathy is closely associated with the Hippo signaling pathway; however, there are still many limitations. At present, it is not completely understood how to modulate the Hippo signaling pathway interaction with metabolism at the cellular level to control systemic metabolism under physiological and pathological conditions, indicating the need for additional studies. It has been reported that renal fibrosis is the result of multiple signaling pathways, such as the Wnt, Notch, and Hippo signaling pathways, and corresponding cytokines, such as TGF-β. Fibrosis of the kidney is a relatively complex process, including signaling pathways interacting with one another. Targeting one signaling pathway or cytokine may not allow the full understanding of the occurrence, development, and prevention of renal fibrosis . The speRecent studies have shown that the Hippo signaling pathway regulates cell proliferation and apoptosis and that it is closely associated with organ structure and function. In addition, the Hippo signaling pathway is involved in the occurrence and development of nephropathy. Recent in-depth studies of signaling pathways have revealed their specificity and diversity in different tissues, cells, and organs. The signaling pathways and molecular mechanisms involved in diseases are complex with various effects resulting from the same molecule occurring in different cells. Thus, the signaling pathways involved in kidney disease need further study to achieve advancements in drug development, clinical diagnosis, and treatment, thereby providing hope for improved treatment of kidney diseases.


All participants provided personal data and were tested for the presence of HTLV-1 and HTLV-2 using enzyme-linked immunosorbent assay and western blotting. HTLV positive samples were selected for Nested-PCR, and viral genotyping by nucleotide sequencing and phylogenetic analysis. HTLV-1 or HTLV-2 infections were detected in 15 families of PWUDs: 27 family members of PWUDs were infected with HTLV-1 (27.8%) and another 20 of them with HTLV-2 (20.6%). Subtypes 1a [subgroup A (54.5%)], 2b (20.5%), and 2c (25.0%) were detected. High horizontal (76.9%) and vertical (61.4%) transmission rates of HTLV were ascertained. Factors that facilitate the acquisition and transmission of HTLV-1 and HTLV-2 were reported by the participants, such as long-term relationships, unprotected sex, breastfeeding, and lack of knowledge about the condition of being a carrier of the retrovirus. Evidence indicates intrafamilial transmission of HTLV from PWUDs to members of their respective families. Key interventions should urgently be employed for the control and prevention of HTLV-1 and HTLV-2 to reduce the spread of this retrovirus in PWUDs and the general population in Northern Brazil and elsewhere.The human T-lymphotropic virus 1 (HTLV-1) and 2 (HTLV-2) can be transmitted between humans by mechanisms associated with horizontal and vertical routes. Recently, high prevalence rates and levels of genetic diversity for HTLV-1 and HTLV-2 were detected among people who use illicit drugs (PWUDs) in the Brazilian state of Pará. None of the PWUDs with HTLV-1 or HTLV-2 were aware of their carrier condition of the retrovirus, and they ability to spread it to their family group, sexual partners, and other contacts. Thus, this study evaluated the presence of HTLV-1 and HTLV-2 in families of PWUDs in the state of Pará, in Northern Brazil. This descriptive study used convenience sampling and accessed 37 PWUDs and their respective families ( The human T-lymphotropic virus (HTLV) belongs to the family Retroviridae, subfamily Oncovirinae, and genus Deltaretrovirus . To dateBased on the nucleotide diversity of its long terminal repeat (LTR) region, there are six molecular subtypes  of HTLV-1 and four  of HTLV-2 . Human mvia transmission from mother to child  and horizontal (unprotected sex between intimate partners) routes . For exaThis descriptive study used convenience sampling and accessed PWUDs and their respective families in 18 municipalities in the state of Pará, northern Brazil . All infIn addition to personal  data assessed, blood samples were collected and tested for HTLV-1 and HTLV-2 infections. The presence of anti-HTLV-1/2 antibodies was evaluated by enzyme-linked immunosorbent assay (EIA) using Murex HTLV-I + II GE80/81 . Non-reactive samples were classified as HTLV-negative, and no additional tests were performed on those samples. EIA-reactive samples were subjected to confirmation by Western blotting (WB) using HTLV Blot 2.4 . All laboratory tests were conducted according to manufacturers’ guidelines.All samples positive for anti-HTLV-1/2 antibodies by EIA and WB were selected for the isolation of nucleic acid using the PureLinkTM Viral RNA/DNA Mini Kit , and then subjected to Nested-PCR for fragment amplification of the 5′ LTR . The PCR1 and AliView  phylogenetic trees were reconstructed with the PhyML 3.1 (3 The sequences obtained in this study were deposited in GenBank (OM835810-OM835889).All sequences were edited and aligned using the BioEdit AliView  programs AliView , obtainehyML 3.1  under thhyML 3.1 . The heuhyML 3.1 . The treIn this study, a set of family members was generated from each of the PWUDs. HTLV-1 or HTLV-2 infection was defined by the presence of antibodies using EIA and WB. The horizontal  and vertical  transmission routes for HTLV-1 and HTLV-2 were considered and highlighted , the majority were men, single, with up to 10 years of education, and low monthly income  and participated in this study. In this sample  presented as members the person who used illicit drugs and his mother, without any indication of a steady sexual partner or children. In total, 119 family members of PWUDs were identified, but only 97 of them were found and participated in the study as described here: mother (n = 27), husband (n = 4), wife (n = 4), steady sexual partner (girlfriend) (n = 5), ex-wife (n = 3), son (n = 15), daughter (n = 16), grandson (n = 11), granddaughter (n = 2), daughter-in-law (n = 5), and son-in-law (n = 5). Ten mothers and two ex-wives of PWUDs had died, and two ex-wives, five daughters, and three sons were not found in the PWUDs’ home municipality. In the sample of family members of PWUDs (n = 97), most were female (63.9%), had up to 10 years of education (72.2%), and had low monthly income (83.5%). The family members’ ages ranged from 1 to 81 years. None of these family members reported: use of illicit drugs in life, exchange of sex for money or drugs during life, and more than one sexual partner in the last 12 months. All PWUDs husbands, wives, and steady sexual partners reported unprotected sex in the last 12 months. PWUDs’ ex-wives haven’t had sex with them for over 24 months.In addition, 37 families of PWUDs were evaluated. Most families (n = 97), 27 family members of PWUDs were infected with HTLV-1 (27.8%) and another 20 of them with HTLV-2 (20.6%) (n = 44). No proviral DNA has been detected in three samples (two sons and a daughter of PWUDs) that tested positive for HTLV-1 using EIA and WB. Subtypes 1a [subgroup A or Transcontinental (54.5%)], 2b (20.5%) and 2c (25.0%) were detected in the proviral DNA samples (Human T-lymphotropic virus 1 or human T-lymphotropic virus 2 infections were detected in 15 families of PWUDs . In tota (20.6%) . No case samples , 2. In Fn = 4), wives (n = 4), ex-wives (n = 3), girlfriend (n = 1), daughters-in-law (n = 4), and sons-in-law (n = 4) were detected with HTLV-1 or HTLV-2, and evidenced the possibility of horizontal transmission, as they reported unprotected sex with infected sexual partners (n = 8), daughters (n = 11), grandson (n = 6), and granddaughters (n = 2) of PWUDs evidenced the possibility of vertical transmission through breastfeeding from infected mother to child  and had up to 10 years of education (66.0%) or were illiterate (25.5%). The age ranged from 1 to 44 years (average age = 21.7 years). All mothers of the PWUDs tested negative for HTLV-1 or HTLV-2. Husbands  and vertical (daughter 1 infected by HTLV-1a through breastfeeding) routes. All family members of PWUDs were unaware that they were infected with HTLV-1 or HTLV-2, and there were no reports of symptoms related to these viral infections. Other schematic representations indicate the spread of HTLV-1 and HTLV-2 in families of the PWUDs in The schematic representations of the PWUD families allowed a clearer view of the entry and spread of HTLV-1 or HTLV-2 in these groups. For example, in This study recorded the presence of HTLV-1 and HTLV-2 among PWUDs and in several members of their families. A high positivity rate of HTLV infection was detected among sexual partners of PWUDs (76.9%), as well as sexual partners of sons and daughters of PWUDs, indicating the importance of the horizontal route for the spread of this virus in the general population. Similarly, a high rate of positivity of HTLV infection was detected in sons, daughters, grandsons, and granddaughters of PWUDs (61.4%), clearly indicating the silent but substantive contribution of the vertical route to the maintenance of high rates of this viral infection, such as prevalence and incidence, by generations. These values are higher than what has been recorded in an epidemiological investigation on mother-child (20.0–21.2%) and spouse-spouse (14.3–51.3%) dyads of patients with HTLV-1 and HTLV-2 treated at the referral center in the metropolitan area of Belém . FurtherIn general, the acquisition and spread of HTLV-1 and HTLV-2 among PWUDs occur mainly through risky sexual contact, and this can be potentiated by different patterns and forms of illicit drug use . The lonThis finding is very important for understanding the epidemiological scenario of HTLV-1 and HTLV-2 in northern Brazil (Amazon region), as it indicates how this retrovirus has been transmitted “silently” for generations. Consequently, there are key interventions to be highlighted for the control and prevention of HTLV-1 and HTLV-2 in this Brazilian region. First, there is a need to provide expanded laboratory testing and surveillance for HTLV-1 and HTLV-2, and clinical guidance and supervision of people living with this retrovirus, especially in municipalities far from the state of Pará’s capital. The concentration of resources, services, and skills techniques exclusively in the metropolitan area of Belém complicates the diagnosis and clinical care of people infected with HTLV-1 and HTLV-2 living in more distant municipalities, such as Almeirim, Óbidos, Melgaço and, Porto de Moz, or rural areas of the state. The precarious transport network and the deficiency of health services in this Brazilian region associated with low education and low economic income of most of the population are obstacles that must be considered and addressed toward generating desirable changes and improvements in the present epidemiological scenario. One suggestion is the establishment of advanced units for diagnosis, guidance, and clinical care of people with HTLV-1, HTLV-2, and other pathogens in microregions within the immense state of Pará. Access to diagnosis is an essential tool for surveillance, prevention, and clinical monitoring, especially in an area with a high rate of infection .Another key intervention is the need for better primary prevention of HTLV infections. Unprotected sex and breastfeeding are important primary risk factors for the spread of this retrovirus. Therefore, education programs aimed at reducing risky sexual behavior must be implemented by public health authorities, including the distribution of resources or prevention materials such as male and female condoms. Yet, the need to better integrate and address HTLV in campaigns for the control and prevention of STIs in the general population should be highlighted. This becomes even more relevant because of the easy viral acquisition and spread in key populations such as PWUDs, who even knowing their HTLV carrier status still engage in unprotected sex and other risky behaviors. The sub-population of pregnant women should be monitored more carefully. Recently, a flowchart for the management of HTLV infection in pregnant women and prevention of mother-to-child transmission has been proposed and showed well-defined measures for monitoring, prevention, and guidance of mothers and neonates in the prenatal, delivery, and post-partum phases . In geneConsidering the duration and intensity of illicit drug use reported by PWUDs in this study, another key intervention is the treatment of drug problems in this particular risk population, including the availability of resources to minimize the harm associated with the use of psychoactive substances. This could reduce the spread of HTLV and other pathogens in the general population, as well as provide better life expectancy and quality of life for PWUDs. Most participants are likely to need treatment for their drug use, which in many instances includes one or multiple drug use disorders  . Mental This study has limitations and should be highlighted. It has a small number of participants, because of the difficulty in accessing the PWUDs, and consequently their families, and the complex logistics for the movement of people and products in the Brazilian state of Pará (Amazon region). The descriptive nature of the study is also a limiting factor. Evidence of intrafamilial transmission of HTLV-1 and HTLV-2 is supported only by phylogenetic findings. Other information and resources could have been accessed and used to support or exclude the possibility of intrafamilial transmission of these retroviruses. A complex suggestion to minimize all these problems is to conduct a prospective study, with a larger sample size, using robust epidemiological methods and accurate laboratory diagnoses for the identification of HTLV-1 and HTLV-2 and, consequently, a more accurate assessment of the possibility of intrafamilial transmission.In summary, this study detected the presence of HTLV-1 and HTLV-2 in PWUDs and several members of their families, as well as documented high rates of transmission of this retrovirus through horizontal  and vertical  routes. Evidence indicates intrafamilial transmission of HTLV subtypes 1a, 2b, and 2c of PWUDs to their respective relatives. Key interventions should urgently be employed for the improved control and prevention of HTLV-1 and HTLV-2 in the state of Pará to reduce the spread of this retrovirus in PWUDs and the general population, represented here by PWUD family members.OM835810-OM835889).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 below: GenBank . The patients/participants provided their written informed consent to participate in this study.AO-F, LCM, JR, BF, and EK: conceptualization. PF, RF, LS, LCM, LFAM, JR, AV, and RI: investigation and methodology. AO-F, PF, and RF: formal analysis. AO-F: writing–original draft and project administration. PF, RF, LS, LFAM, AV, RI, BF, and EK: writing–review and editing. AO-F, LCM, JR, and BF: funding acquisition. All authors contributed to the development of research, contributed to the article, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.