In the last years, proteomics has represented a valuable approach to elucidate key aspects in the regulation of type I/III interferons (IFNs) and autophagy, two main processes involved in the response to viral infection, to unveil the molecular strategies that viruses have evolved to counteract these processes. Besides their main metabolic roles, mitochondria are well recognized as pivotal organelles in controlling signaling pathways essential to restrain viral infections. In particular, a major role in antiviral defense is played by mitochondrial antiviral signaling (MAVS) protein, an adaptor protein that coordinates the activation of IFN inducing pathways and autophagy at the mitochondrial level. Here, we provide an overview of how mass spectrometry-based studies of protein–protein interactions and post-translational modifications (PTMs) have fostered our understanding of the molecular mechanisms that control the mitochondria-mediated antiviral immunity. Mitochondria are the powerhouse of the cell due to their primary contribution in cell respiration. In addition to their canonical role in cellular metabolism, mitochondria have emerged as a central platform for the regulation of antiviral signaling pathways . AlthougIn the last years, remarkable advances have been made in our knowledge about the molecular mechanisms underlying MAVS signaling. The development of innovative technologies in the field of transcriptomics, proteomics, reverse genetics, and structural and cell biology has dramatically improved our understanding of how RNA viruses can be sensed and lead to the expression of hundreds of genes dedicated to infection inhibition. In particular, yeast two-hybrid and mass spectrometry (MS) approaches have provided detailed information on how MAVS-dependent antiviral responses are rapidly activated, and also tightly controlled by protein catabolic processes, i.e. the ubiquitin–proteasome system and autophagy, to avoid excessive inflammation and cell death. Moreover, these approaches have widened our understanding of how viral proteins are able to inhibit MAVS activation to accomplish viral replication and how energy metabolism may influence mitochondrial antiviral pathways.Here, we review our current view of MAVS signaling, highlighting how proteomics has deepened our understanding of this pathway regulation in terms of protein–protein interactions and post-translational modifications (PTMs).The innate immune response is the first line of defense against invading pathogens that is activated following the recognition of specific entities, called pathogen-associated molecular patterns (PAMPs), through a series of receptors, termed pattern recognition receptors (PRRs). Upon detection of PAMPs, the transcription of a myriad of antiviral genes is activated, establishing a cellular antiviral state that helps cells to restrict and/or clear infection .Based on their structures, locations, and functional specificities, PRRs are separated into discrete families, which include the membrane bound Toll-like receptors (TLRs), the cytosolic nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), cyclic GMP-AMP (cGAMP) synthase (cGAS), and retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) . The actThe TLRs are a family of transmembrane PRRs that are activated by the binding of ligands to their C-terminal leucine-rich repeats. Ten TLRs have been identified in humans, with TLRs 1, 2, 4, 5, 6, and 10 located at the cell surface and TRLs 3, 7, 8, and 9 spanning the endosomal membrane. Plasma membrane TLRs mainly recognize microbial wall components, such as lipids, polysaccharides, and proteins, while intracellular TLRs sense pathogen nucleic acids . StimulaMitochondria are directly involved in the regulation of TLR activity. A relevant example is represented by TLR1, TLR2, and TLR4, which were reported to enhance mitochondrial ROS generation in bacteria-infected macrophages to potentiate the antimicrobial killing . This efThe NLR family of PRRs are cytoplasmic proteins that stimulate innate immunity in response to both PAMPs, such as microbial components , and damage-associated molecular patterns (DAMPs), including host components , and environmental sources  . Upon acA direct link between mitochondria and NLR activation has been reported for NLRP3. Active NLRP3 translocates from cytosol to mitochondria, where it functions as a scaffold for inflammasome assembly . Here, NCyclic GMP-AMP synthase is one of the cellular sensors of non-self DNA, together with the endosomal TLR9 and the NLR family protein Absent in Melanoma 2 (AIM2). Cytosolic DNA binds to cGAS and stimulates its enzymatic activity to form the second messenger cGAMP . cGAMP bAlthough DNA and RNA sensing responses occur through independent receptors, the induction of the antiviral response by one of these agents also relies on the activity of the other pathway. Multiple molecular mechanisms are responsible for this strict interdependence, including the direct interaction of the main regulators of these pathways at the ER–mitochondria contact sites, as well as the fact that many downstream factors are shared between these pathways. For a more exhaustive review of the crosstalk between RNA and DNA sensing pathways, see Apart from the endosome localized TLRs, the recognition of non-self RNA is mediated by the cytosolic sensors of the RLR pathway .The RLR family contains three members: RIG-I, melanoma differentiation associated gene 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2). All RLRs are characterized by a central DEAD box helicase/ATPase domain and a C-terminal regulatory domain (CTD), necessary for RNA binding and to prevent constitutive activation .Moreover, RIG-I and MDA5 possess two N-terminal CARDs that mediate their oligomerization following RNA binding and are required for the activation of the downstream adaptor protein MAVS . RIG-I aThe adaptor protein MAVS  is essential to drive innate immunity in response to RNA virus infection . MAVS isMitochondrial antiviral signaling-dependent antiviral signaling initiates after RIG-I and MDA5 sense viral RNA in infected cells . In the Interestingly, the spontaneous aggregation of MAVS in the absence of viral RNA is prevented by multiple N-terminal truncated proteins that are encoded by the MAVS transcript . In addiOnce recruited to the MAVS signalosome, TRAF proteins stimulate two molecular cascades leading to: (a) the phosphorylation of the transcription factors IRF3 and IRF7 by TBK1 and IKKε to induce the expression of type I/III IFNs and (b) the phosphorylation of the transcription factor NF-κB by the IKKα/β/γ complex to upregulate the expression of inflammatory genes . In turnMass spectrometry-based analyses combined to site-specific mutagenesis approaches have greatly contributed to understand how PTM controls MAVS antiviral response . This haThe main PTM responsible for the regulation of RIG-I, MDA5, and MAVS activity is ubiquitination, which acts as either a degradative or regulatory signal, depending on the type of polyubiquitin chain that is formed .A series of E3 ubiquitin ligases catalyzing non-degradative lysine 63 (K63)-linked ubiquitination, including TRIM25, RIPLET, TRIM4, and MEX3C, were reported to induce MAVS activity by triggering ubiquitin-dependent RIG-I tetramerization through their CARDs . InteresK63-linked ubiquitination is also necessary for the oligomerization of MDA5. TRIM65 was identified by an LC-MS/MS-based interactome analysis as the E3 ligase responsible for this regulation by targeting the helicase domain of MDA5 at lysine 743 . Using aRetinoic acid-inducible gene I/MDA5-mediated activation of MAVS needs to be tightly regulated in order to ensure the optimal activation and timely termination of innate antiviral response. To this aim, K63-linked ubiquitination of RIG-I and MDA5 is countered by a series of deubiquitinating enzymes (DUBs), such as USP3, USP21, and CYLD . InsightDegradative ubiquitination also contributes to turning off the mitochondrial antiviral response by regulating the stability of RIG-I and MDA5. The E3 ligases STUB1, RNF122, RNF125, TRIM13, and TRIM40  were shown to mediate lysine 27 (K27)- or lysine 48 (K48)-linked polyubiquitination of RIG-I/MDA5, which results in their proteasomal degradation . How degRetinoic acid-inducible gene I stability is also controlled by autophagy, a catabolic process that plays an important role in the regulation of innate immune response by both degrading invading pathogens and contributing to the termination of the inflammatory response . The autConversely, degradative ubiquitination of MDA5 and RIG-I is negatively regulated by different mechanisms, such as deubiquitination, which is mediated by the DUB USP4 , and sumRecently, NLRP12, a protein of the NLR family, has been reported to negatively control the RLR signaling pathway by modulating both K63- and K48-linked ubiquitination of RIG-I, uncovering an important crosstalk between these innate immune receptor families. In detail, NLRP12 interacts with the ubiquitin ligase TRIM25, preventing RIG-I activation mediated by K63-linked ubiquitination, as well as with RNF125, enhancing K48-linked degradative ubiquitination of RIG-I . Of noteMitochondrial antiviral signaling is a direct target of both non-degradative and degradative ubiquitination in the course of antiviral response .N-acetylglucosamine (UDP-GlcNAc) is linked by the O-GlcNAc transferase. Moreover, metabolomic analysis of viral infected macrophages revealed an increase of intermediate metabolites involved in hexosamine biosynthesis, the pathway responsible for the generation of UDP-GlcNAc. The functional relationship between RLRs and glucose metabolism was confirmed by the observation that lactate, the end product of glycolysis in anaerobic conditions, acts a negative regulator of MAVS signaling by preventing RIG-I–MAVS complex formation . The PLK1–MAVS interaction, identified through a yeast two-hybrid screen, interferes with the binding of MAVS with TRAF3 and attenuates IFN signaling during viral infection as well as during the G2/M phase of the cell cycle . RecentlA third type of PTM that has been shown to regulate MAVS signaling is protein acetylation. LC-MS/MS analysis identified lysine residues 858 and 909 as acetylated residues of RIG-I that contribute to preventing its oligomerization in resting conditions. The acetyltransferases responsible for this PTM remain to be characterized, while HDAC6 was identified as the deacetylase required for removal of these acetyl groups during viral infection to allow RIG-I activation .In addition to PTMs, proteomic analyses have provided a large set of information on how MAVS signaling is regulated by direct protein–protein interactions with cellular and viral factors . InteracVarious studies have reported that ISGs that are induced upon MAVS activation may establish a positive-feedback loop to potentiate the antiviral signaling at multiple steps . For exaThe cytoskeletal network is another source of proteins that were found to stimulate the antiviral response by interacting with components of MAVS signaling. Focal adhesion kinase (FAK) is an actin-associated tyrosine kinase that binds to MAVS in a viral infection-dependent manner and potentiates its signaling independently of its kinase activity . Two micA recent study in which global RNA interference analyses were combined to experimentally and computationally derive interactome data has provided an in-depth view of the RIG-I protein interaction network and identified new processes that influence the host response to viral RNA, including the unfolded protein response, WNT/β-catenin signaling, and RNA metabolism. The role of K-Homology Splicing Regulatory Protein (KHSRP), one of the best hits obtained from this screening, was further characterized, showing that its interaction with RIG-I is required to maintain the receptor in an inactive state in resting conditions .The mitochondrial resident factors NLRX1, LRPPRC, MFN1, MFN2, TOM70, PHB1/2, and LONP1 have been described to interact with MAVS and regulate the antiviral signaling.NLRX1 (nucleotide-binding domain and leucine-rich repeat containing family member) was the first mitochondrial protein identified as a negative regulator of MAVS . NLRX1 iLRPPRC, a protein known to regulate mitochondrial RNA stability, has been characterized as a negative regulator of the mitochondrial-mediated antiviral immunity during HCV infection. In particular, LRPPRC interacts with MAVS and inhibits its signaling by preventing the association with TRAF3 and TRAF6 .The mitochondrial fusion protein MFN2 has been identified as a negative regulator of MAVS-mediated antiviral signaling through an LC-MS/MS-based interactome analysis. MFN2 binds to MAVS, preventing its oligomerization and the propagation of the downstream antiviral response . This MFAnalysis of MAVS interacting proteins upon mitochondrial enrichment and GeLC-MS/MS identified the mitochondrial protein TOM70 (translocase of outer membrane 70) as a novel component of the MAVS signalosome. In detail, Tom70 acts as a promoting factor of the antiviral response by functioning as a scaffold for the recruitment of both IRF3 and TBK1, in association with HSP90, to MAVS .A positive role of the inner mitochondrial membrane (IMM) proteins PROHIBITIN 1 (PHB1) and PHB2 in MAVS signaling has recently emerged in a proteomic study aimed at characterizing the function of the coiled coil domains of these mitochondrial scaffold proteins. Upon viral infection, PHBs were shown to form a complex with MAVS oligomers, which also includes the AAA(+) ATPase proteins Caseinolytic Peptidase B (CLPB) Homolog and ATPase Family AAA Domain Containing 3A (ATAD3A), thus establishing a bridge between the OMM and IMM that is required for the efficient activation of the RIG-I signaling pathway .The mitochondrial protease LONP1 has been identified as a positive regulator of MAVS in a coIP/MS-based analysis from purified MAM, a study that was performed using surfactants hydrolyzable at low pH to reduce detergent interference in MS . HoweverMitochondrial antiviral signaling activity is also regulated by autophagy through protein–protein interaction. MAVS directly binds to the autophagosome protein LC3 through an LC3-interaction region (LIR) at the MAVS N-terminal region. Through this interaction, MAVS was reported to function as a mitophagy receptor for the engulfment of mitochondria that are damaged upon excessive activation of antiviral signaling . MoreoveThe identification of gC1qR (receptor for globular head domain of complement component C1q) and Sorting nexin 8 (SNX8) as MAVS interacting proteins that regulate the antiviral response suggests a role of other signaling pathways to MAVS activity that remain to be contextualized .Viruses have evolved different mechanisms to inhibit MAVS activity as part of their immune evasion strategies. Viral proteins are able to interfere with the binding of MAVS to the upstream RLR sensors. The non-structural protein NS4A of Dengue and Zika viruses binds to the CARD of MAVS, preventing RIG-I/MDA5 accessibility and hampering RLR signal transduction . Zika NSn-dodecyl β-D-maltoside as a detergent to preserve the integrity of mitochondrial membrane complexes. We found that HCV NS5A interacts with the mitochondrial protein LRPPRC and represses the antiviral response by promoting LRPPRC association with MAVS, which results in a reduced association of MAVS with TRAF proteins  in dendritic cells. In these cells, the RNA helicase DDX3 acts as an intracellular sensor of abortive HIV-1 RNAs that stimulates MAVS activity by promoting its interaction with TRAF3. HIV-1 is able to block this pathway through the HIV-1 envelope protein GP120, which, by binding to the C-type lectin receptor DC-SIGN, activates PLK1 to interfere with the formation of MAVS–TRAF3 complex .Finally, a large series of viral proteases induces the proteolytic cleavage of MAVS to release it from the outer membrane of mitochondria. These include: HCV NS3/4A viral protease, which cleaves MAVS at cysteine 508 ; hepatitThe mitochondrial proteome is a highly dynamic entity that undergoes profound changes in response to a variety of stress conditions in the attempt to mount an adaptive response or, when not feasible, to trigger programmed cell death. In this review, we discussed recent findings describing how the host cell response to RNA virus infection is centered on the mitochondrial outer membrane. In this subcellular compartment, MAVS signalosome is rapidly assembled to recruit key components of IRFs and NF-κB signaling pathways and induces the expression of hundreds of genes with antiviral properties. In particular, we highlighted how proteomic approaches have contributed to the characterization of protein networks and their PTMs that regulate MAVS signaling. In addition, for its unbiased characteristics, proteomics has provided several new insights on the crosstalk of the mitochondrial antiviral response with other PPRs, such as NLR and lectins, or other cellular processes, such as glucose metabolism. The key contribution of proteomic studies to unraveling the complexity of the antiviral response is expected to have an important impact on the development of novel therapeutic strategies aimed at suppressing viral infection and enhancing immune responses.GR, GF, and FC wrote the text and prepared the figures. TV and MP contributed to literature search and revised the manuscript and discussion.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.


Thymus vulgaris honey (TVH) and Origanum vulgare essential oil (OVEO) and their mixture effect on carbon tetrachloride (CCl4)-induced toxicity.The study investigated the chemical composition, antioxidant content, and antioxidant activity of 4-induced toxicity. Intraperitoneal injection of CCl4 (1 mL/100 g) was used to induce toxicity. The doses of TVH and OVEO were 1 mg/kg.b.wt, and 50 mg/kg.b.wt, respectively. HEM contains TVH (1 mg/kg.b.wt) and OVEO (50 mg/kg.b.wt).The study conducted physicochemical characterization and chemical analysis of TVH and OVEO with the use of gas chromatography–mass spectrometry and high-performance liquid chromatograph (HPLC). The antioxidant activity of TVH and OVEO was done with the use of 1,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. The study used nine groups of rats to investigate the effect of TVH, OVEO, and a mixture of TVH and OVEO (HEM) on CClst time, epicatechin gallate, and at a high concentration. OVEO includes a high percentage of carvacrol and thymol. With the use of DPPH, OVEO was more potent than TVH. CCl4 caused significant liver and kidney damage and lipid disorders, which were alleviated by HVT, OVEO, and HEM. HVT was more potent than OVEO (p<0.05), and HEM was more potent than HVT and OVEO (p<0.05).TVH has a high content of phenols, flavonoids, and flavanols. HPLC analysis showed that TVH contains, for the 1st time in TVH, and OVEO contains a high percentage of thymol and carvacrol. Epicatechin gallate might be useful as a marker for TVH. Mixing OVEO and TVH significantly potentiated their protection against CCl4-induced liver and kidney toxicity.The study identified high content of epicatechin gallate for the 1 Thymus vulgaris honey (TVH) and Origanum vulgar essential oil (OVEO) have important biological activities and have great potential to be introduced in the illness treatment. TVH is unifloral honey, which has antimicrobial activity and wound healing properties. It is useful in managing radiation-induced mucositis and xerostomia in head and neck cancer patients [And your LORD taught the bee to build its cells in the mountains, on the trees, and in men’s habitations, then to eat of all the fruits of the earth and find with skill the spacious paths of its LORD, their issues from within their bellies, a drink of varying colors, wherein is healing for men, verily in this is a sign for those who give thought). O. vulgar is one of the species of Origanum. Herbs of this family are used to improve the flavor of foods. A recent review showed that O vulgar has an anticancer, anti-inflammatory, antiproliferative, antibacterial, and hepatoprotective effect [4) is a volatile organic alkyl halogen, which is metabolized in the liver and generates trichloromethyl (CCl3) and trichloromethyl peroxyl (CCl3O2).A large amount of data showed that honey has biological activities such as antimicrobial, anti-inflammatory, antioxidant, and immune-modulatory activities -3. Thymupatients -6. Honeye effect . The kid4 is very toxic to the liver and kidney and used experimentally to induce liver and kidney damage. Others and we have found that honey has a protective effect against CCl4-induced liver and kidney toxicity [O. vulgare leaves extract against CCl4-induced hepatotoxicity in rats has been reported [4-induced liver and kidney toxicity. Therefore, the present study examined the protective effect of TVH, OVOE, and their combination (HEM) against CCl4-induced toxicity in rats. It was well known that the chemical composition of various honey samples is different [CCltoxicity -10. Furttoxicity ,12. The reported . Howeverifferent ,15. Ther4-induced liver and kidney damage.The study showed for the first time the presence of epicatechin gallate in TVH collected in Fez, Boulemane, Morocco, and the ability of TVH, OVEO, and HEM to protect against CClThe ethical institutional committee, Faculty of Sciences Dhar El Mahraz, University Sidi Mohamed Ben Abdallah, Fez, Morocco, approved the protocol. All the experimental proceedings achieved in laboratory animals followed the internationally accepted standard guidelines for animal care. The authors tried to minimize animal suffering and the number of animals used.This study was conducted in June month of 2017 at Sidi Mohamed Ben Abdellah University, Morocco.T. vulgaris, Zaitra; Arabic name) from beekeepers, Fez, Boulemane, Morocco. The honey sample was collected in 2015 and stored at room temperature (22-24°C) until analysis.The authors purchased honey  radical scavenging activity according to the method by Brand-Williams et al. . Briefly(The absorbance of negative control - The absorbance of the sample)/the absorbance of negative control)×100.50 was determined .Based on each sample, ICThe honey sample was subjected to base hydrolysis and extracted with ethyl acetate (liquid-liquid extraction) described by Aljadi and Yousoff . In brieRegarding GC/MS analysis, the OVOE sample was diluted in hexane with a dilution of 10:100. The analysis of volatile constituents was completed using GC/MS method , equipped with an HP-5MS column nonpolar fused silica . The temperature of the injector was 250°C. The column temperature was programmed from 40 to 260°C at 2°C/min. The carrier gas was helium, the flow rate was 1 mL/min, and the volume of sample injected was 1 μL of diluted oil. The identification of the components has been made by determination of their indices of retention Kovats index (Ki) compared to a Ki of a series of n-alkanes (C8-C20) and by comparing their mass spectra recorded with those stored in the database of the spectrometer , and the literature [Forty-five adult male Wister rats (221±22 g) were used. The animals were kept in a diffusely lit and temperature-controlled room with a diurnal 12 h light cycle, where the temperature (25±1°C) and relative humidity (55±5%) were kept constant. The animals were given free access to a standard laboratory diet and water before the experiments.OVEO was dissolved in tween 80, and TVH was dissolved in water, and they mixed with each other. One mL of HEM containing TVH (1 g/kg.b.wt) and OVEO (50 mg/kg.b.wt) was used and delivered to the animals by gavage.4 (1 mL/100 g.b.wt) dissolved in olive oil, 10% solution, twice per week for 2 weeks. The administration of TVH, OVEO, and HEM was started on the 1st day of CCl4 administration and was continued for 2 weeks.The experimental animals received intraperitoneal injections of CClThe animals were randomly divided into nine groups, five animals each, as follows:Group I (water): The animals received drinking water 1 mL/kg.b.wt every day by gavageGroup II (Tween 80): The animals received Tween 80 (2%) by gavage (1 mL/kg.b.wt) every dayGroup III (TVH): The animals received TVH by gavage (1 g/kg.b.wt) every dayGroup IV (OVEO): The animals received OVEO by gavage (50 mg/ kg.b.wt) every dayGroup V (HEM): The animals received HEM 1 mL/kg.b.wt by gavage every day4): The animals received CCl4Group VI (CCl4+ TVH): The animals received CCl4 and treated with TVH by gavage at a dose of 1 g/kg.b.wt every dayGroup VII (CCl4 + OVEO): The animals received CCl4 and treated with OVEO by gavage at a dose of 50 mg /kg.b.wt every dayGroup VIII (CClGroup IX (CCl4 + HEM): The animals received CCl4 and treated with HEM by gavage every day.The doses of TVH, OVEO, and Tween 80 were used according to the previous publications ,26,27. A<0.05 was considered significant.Results were expressed as mean ± SD. Statistical analysis was carried out by ANOVA with the use of GraphPad Prism  6.0 program. pThe physicochemical parameters of honey showed that the pH was 3.90±0.01; free acidity was 37.93±2.25 mEq/kg, moisture was 16.4±0.01%, electric conductivity was 449±1731 mS/cm, ash was 0.24±0.01 %, and melanoidin was 1.10±0.02. The Pfund scale value was 0.33±0.01, which indicated that the color was dark amber.Mineral content analysis of honey showed that the concentration of potassium was 829.55±0.51 mg/kg, sodium was 256.32 ± 0.15 mg/kg, magnesium was 61.47±0.21 mg/kg, and calcium was 166.22±0.09 mg/kg. OVEO analysis showed that the pH was 5.02, density was 0.9484 g/mL, and the refractive index was 1.5153.The phenol content value was 74.05±1.21 mg AGE/100 g, flavonoids content was 59.34±1.08 mg Eq Q/100g, and flavonol was 15.08±0.04 mg Eq Q/100 g.The honey was analyzed through HPLC under the same chromatographic conditions . The ana50 amount of OVEO was 0.30±0.02 mg/mL and of TVH was 10.85±0.02, which means that the DPPH scavenging activity of OVEO was higher than that of TVH (p<0.05).The total antioxidant capacity of TVH was 163.51±8.55 mg Eq AA/g. The IC50 value of BHT was 0.009±0.0001 mg/mL in the DPPH assay. However, the IC4-untreated rats is demonstrated in The effect of TVH, OVOE, and HEM on kidney and liver function and lipid profile in CCl4-treated rats, CCl4 caused a significant elevation of AST, ALT, AP, and total and direct bilirubin, and caused a significant lowering of total protein (p<0.05)  . TVH, OV4 significantly increased all the lipid parameters and decreased HDL significantly. However, TVH, OVOE, and HEM significantly lowered the elevated lipid parameters as well as elevated the lowered HDL. TVH was more potent than OVOE, and HEM was more potent than TVH and OVOE (p<0.05).TVH was more potent than OVOE, and HEM was more potent than TVH and OVOE (p<0.05). Lipid assays revealed that CCl4 induced liver and kidney damage. TVH was more potent than OVOE to protect against CCl4 toxicity, and HEM was more potent than TVH and OVOE individually.The data presented here demonstrated for the first time the presence of epicatechin gallate in Moroccan TVH and the protective effect of TVH, OVEO, and their combination against CClCeratonia siliqua honey samples (range from 4.17 to 5.05) collected from Morocco [The pH of TVH was 3.90±0.01, which was less than the pH of  Morocco . Further Morocco . It mean Morocco -32. The C. siliqua honey collected from Morocco [Other compositions, including water content, ash, and electrical conductivity, are almost within the range found in  Morocco .The ash content and electrical conductivity correlated with the mineral content of honey . The ashThe electrical conductivity of TVH was 449±1.73 mS/cm. According to the EU, the maximum limit value of electrical conductivity for thyme honey is 800 μS/cm . The eleThe honey moisture content is an important criterion to determine the quality of honey. The water content depends on nectar, storage and environmental conditions, and harvest season. TVH moisture was 16.4±0.01%, and it was within the range of Iranian honey moisture (13.97-17%), Portuguese honey moisture (13.1 and 16.9), and within limits (≤20%) set by the EU ,32,34,35The mineral content is an essential part of the nutritional values of honey. The results showed that TVH contains necessary minerals that involve in several vital functions of the animals and the human body. It was found that potassium was the most abundant element in the thyme honey. The range of potassium concentration in TVH is almost within the range obtained in other honey types collected from different countries ,36. AnotThe Pfund scale value of TVH was 0.33± 0.01, which indicated that the color was dark amber. The color is similar to carob honey and Portuguese honey ,37. The Phenolic compounds are plant-derived secondary metabolites and transferred through the nectar to the honey. The phenolic compounds can be classified into phenolic acids and flavonoids. The content of phenolic compounds of honey depends on the floral and geographical origins .The value of phenol content in TVH was 74.05±1.21 mg GAE/100 g. It was found that phenol content of 17 commercial honey samples from Morocco varied from 16.38 mg GAE/100g in citrus honey to 92.37 mg GAE/100 in thyme honey from Rachidia . TherefoWith the use of HPLC, the standard compounds were detected at 280 nm because most of the phenolic compounds showed reasonably high absorbance at this value. Five phenolic compounds were identified in TVH extract: Ferulic acid, gallic acid, caffeic acid, epicatechin gallate, and pyrogallol. The major constituent in the sample was epicatechin gallate, with a concentration of 6.91±0.05 mg/100 g. Ferulic acid, gallic acid, and caffeic acid have been reported in Malaysian honey . Twenty-Epicatechin gallate was detected for the first time in TVH. Epicatechin gallate was not detected in honey before. It is useful in treating wounds, and it exhibits a potent anti-inflammatory and analgesic effect by lowering levels of plasma prostaglandin E2, tumor necrosis factor-α, interleukin-1β (IL-1β), and IL-6 -44. TherThe value of flavonoid content was 59.34±1.08 mg QE/100 g, and flavonol was 15.08±0.04 mg QE/100 g. Flavonoid content was 0.42 mg QE/100g in citrus honey and 13.962 mg QE/100g in black cumin honey . Therefo50 values of thyme honey from Rachidia, Morocco, were 52.85±1.2 mg/mL, and from Saouira, Morocco, were 51.76±1.26 mg/mL [50 values of 21 different types of honey, which were derived from the Olympus mountain area, Greek, ranged from 7.5 to 109.0 mg/mL, while Manuka honey exhibited a weak antioxidant activity  in DPPH assay [50 of Malaysian Tualang, Gelam, and Borneo tropical honey ranged between 5.24±0.40 and 17.51±0.51 mg/mL, and therefore, the antioxidant activity of TVH lies in the same range [50=24.5 mg/mL, which is weaker than TVH as an antioxidant. The use of DPPH to evaluate the antioxidant activity is based on determining the ability of TVH to act as a hydrogen or electron donor in the conversion of DPPH in its reduced form (DPPH-H).The total antioxidant capacity of TVH was 163.51±8.55 mg Eq AA/g. The IC26 mg/mL . This mePH assay . Therefome range . JordaniO. vulgare from Morocco and other countries, which showed that the major compound was carvacrol [The GC/MS analysis of OVOE revealed two major compounds: Carvacrol 48.38%) and thymol (26.55%), which agrees with another study . Furtherarvacrol ,46. The arvacrol . The difarvacrol . It has .38% and arvacrol .50 value of OVEO prepared from the aerial part (stem and leaves) of the plant was 0.30±0.02 mg/mL. It is almost similar to the results of the IC50 values of the Chinese OVEO prepared from leaves/flower (0.332±0.040 mg/mL) and the plant root ( 0.357±0.031 mg/mL), but less than IC50 values of essential oil prepared from the plant stem (0.501±0.029 mg/mL) [50 of OVEO ranged from 0.32 mg/mL to 0.76 mg/mL, depending on incubation time [The IC9 mg/mL) . It was ion time . However4 significantly increases serum levels of AST, ALT, and ALP compared to the control group. However, administration of TVH, OVEO, or their combination to the animals with CCl4-induced hepatotoxicity decreased significantly liver enzymes compared to the CCl4 treated group. This finding showed the hepatoprotective effect of TVH, OVOE, and HEM. Interestingly, HEM showed a more profound hepatoprotective effect than TVH or OVEO. The potentiation of HEM effect might be due to the synergistic activity of natural ingredients in TVH and OVEO. It was found that O. vulgare leaf extract has significant protection against CCl4-induced hepatotoxicity in a dose-dependent manner. It lowered serum ALT, ALP, and AST levels and elevated Glutathione S-transferase, catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase, and GSH levels in liver tissue [4 intoxication by normalizing liver enzymes and kidney function [The results showed that CClr tissue . Furtherfunction -10.4 significantly increased TG levels, cholesterol, and LDL and decreased serum levels of HDL compared to the control group. TVH, OVEO, and HEM markedly ameliorated these effects. This finding agrees with the impact of Sidr honey on lipid profile in CCl4 toxicity [4-untreated rats, TVH or OVEO has no significant effect on lipid profile; however, their combination caused a considerably lower TC, LDL, and VLDL and considerable HDL elevation. This result is most likely due to an additive or synergistic effect of active ingredients in TVH and OVEO. This finding makes their combination a good candidate for prophylaxis of dyslipidemia and cardiovascular diseases.CCltoxicity . In CCl44 significantly increased the blood level of creatinine and urea. The mechanism of CCl4 induced renal toxicity is almost the same as that of the liver. Furthermore, CCl4 has a strong affinity to the kidney cortex because it contains a high level of cytochrome P-450 [4 causes a significant elevation of oxidative stress markers in the blood and kidney tissues, which were markedly alleviated by Carob honey’s administration (Unpublished).CClme P-450 . The adm4-treated rats [O. vulgare, T. vulgaris, and other plants. A recent review showed that carvacrol has antioxidants, antimicrobial, and anti-inflammatory activity [TVH has a high content of epicatechin galatte and ferulic acid, while OVOE contains carvacrol and thymol. Epicatechin gallate has anti-inflammatory, analgesic, and wound healing properties ,43. Feruted rats . Carvacractivity . The mec4-induced liver and kidney damage, electrolyte disturbances, and lipid disorder. Although the ability of OVEO to scavenger DPPH was higher than TVH; TVH was more potent than OVEO to protect against CCl4 toxicity. Therefore, TVH, OVEO, and HEM are significant interventions to protect against chemical toxicity and might be useful in other causes of kidney and liver failure and dyslipidemia. While TVH and OVEO did not significantly affect kidney and liver function in CCl4 non-treated rats, their combination causes a substantial improvement in lipid profile and kidney and liver function. Therefore, the use of HEM in normal individuals might improve the lipid profile and liver and kidney function and protect against cardiovascular disease. These findings are exciting and will stimulate further preclinical and clinical studies in this growing field.The main finding of this study is summarized in HI, AAb, AAbd, and BL designed the experimental protocols and participated in the practical work and writing of the paper. NA analyzed the data and results and wrote the manuscript for publication. TA did the statistical analysis. All authors read and approved the final manuscript.


Affecting more than 26 million people worldwide and with rising prevalence, heart failure (HF) represents a major global health problem. Hence, further research is needed in order to abate poor HF outcomes and mitigate significant expenses that burden health care systems. Based on available data, experts agree that there is an urgent need for a cost-effective prognostic biomarker in HF. Although a significant number of biomarkers have already been investigated in this setting, the clinical utility of adding biomarker evaluation to routine HF care still remains ambiguous. Specifically, in this review we focused on uric acid (UA), a purine metabolism detriment whose role as cardiovascular risk factor has been exhaustingly debated for decades. Multiple large population studies indicate that UA is an independent predictor of mortality in acute and chronic HF, making it a significant prognostic factor in both settings. High serum levels have been also associated with an increased incidence of HF, thus expanding the clinical utility of UA. Importantly, emerging data suggests that UA is also implicated in the pathogenesis of HF, which sheds light on UA as a feasible therapeutic target. Although to date clinical studies have not been able to prove the benefits of xanthine oxidase in HF patients, we discuss the putative role of UA and xanthine oxidase in the pathophysiology of HF as a therapeutic target. With a worse prognosis than breast cancer in women and bladder cancer in men, heart failure (HF) represents a major global health problem ,2. This Owing to limited clinical applicability and low precise risk stratification of HF prognostic biomarkers, further research is needed in order to abate poor HF outcomes and mitigate significant expenses that burden health care systems. Specifically, in this review we focused on uric acid (UA), a purine metabolism detriment whose role as a cardiovascular risk factor has been exhaustingly debated for decades . Apart fVirtually any disease or defect that impairs heart structure or function can subsequently lead to HF development. Although most commonly caused by coronary artery disease (CAD), unregulated diabetes, and hypertension, a palette of other etiologic factors, both intra- and extracardiac, can induce HF development . In multiple pathophysiological pathways that are operative in HF, such as myocardial necrosis, upregulation of the renin-angiotensin-aldosterone system (RAAS), overt activation of the sympathetic nervous system, and endothelial dysfunction, a recently recognized pathologic process of endothelial-to-mesenchymal transition (EndoMT) emerged as a potent pathobiological driver of pro-fibrotic signaling pathways in HF, thus leading to myocardial fibrosis and adverse ventricular remodeling ,21,22,23In 2013, Paulus and Tschöpe elaborated a pathophysiological model of HF with preserved ejection fraction (HFpEF), which proposes that highly prevalent co-morbidities of HF such as ageing, diabetes mellitus, metabolic syndrome, salt-sensitive hypertension, atrial fibrillation (AF), anemia, chronic obstructive pulmonary disease, and especially obesity exert their detrimental effects on the heart via endothelium of coronary microcirculation, which, as hypothesized, acts as a sort of central processing unit and transfers damage to the heart . AccordiUA is the end product of both dietary and endogenous purine metabolism in humans . Due to Nevertheless, the biological effects of UA regarding oxidative stress are rather confounding. Unlike the antioxidant effects that UA exerts in extracellular, hydrophilic milieu, intracellularly it imposes detrimental effects, acting as a pro-oxidant . Multipl2 max and a positive correlation between UA levels and minute ventilation/carbon dioxide production (VE/VCO2), both of which suggest that increased SUA concentrations may reflect an impairment of the oxidative metabolism with consequent exercise intolerance in HF [The last two steps of purine metabolism are catalyzed with xanthine oxidase (XO) . The mence in HF . Apart fce in HF ,70,71. Xce in HF . Finallyce in HF . It has ce in HF ,75. In cce in HF . InteresIn the HF setting, elevated SUA levels are owed to at least two distinct mechanisms . The firn = 10) and the fact that the use of diuretics, important regulators of UA excretion, was not clearly defined in the individual studies. The combination of UA and N-terminal pro-brain natriuretic peptide (NT-ProBNP) levels appears to be even more useful in AHF prognosis, as Park et al. demonstrated that a combination of the two is a better independent predictor for short-term outcomes in this setting than either of the markers alone [P < 0.001) for death [To date, myriads of studies have been conducted regarding the clinical significance of UA in both AHF and CHF. A recent meta-analysis suggested that high SUA levels independently predicted all-cause mortality of patients with AHF, with hyperuricemia being associated with a 43% increase in all-cause mortality . The samrs alone . In our or death . Thus, ior death . Overallor death . In lineor death .About half of the patients suffering from HF with either preserved or reduced ejection fraction EF exhibit SUA concentrations above the upper reference limit ,97. The The most widely discussed aspect of the role of UA in HF is in regard to the prognosis of patients with CHF. An important notion concerning prognostic studies is that older studies (before 2010) rarely adjusted variables relevant to the association of UA and adverse outcomes of HF patients . The afoBased on the evident pathophysiologic role of XO and UA in HF development, multiple authors tested the therapeutic potential of XO inhibition/UA reduction . A panelBased on the available data it seems that UA has a wide set of feasible routine clinical applications in HF . In AHF,


S-enantiomer of CE-123 ((S)-CE-123) and R-modafinil in cognitive- and reward-related brain areas of adult male rats. In vivo single unit recordings in anesthetized animals showed that (S)-CE-123, but not R-modafinil, dose-dependently (1.25 to 10 mg/kg i.v.) reduced firing of pyramidal neurons in the infralimbic/prelimbic (IL/PrL) cortex. Neither compound the affected firing activity of ventral tegmental area dopamine cells. In freely moving animals, (S)-CE-123 (10 mg/kg i.p.) increased extracellular dopamine levels in the IL/PrL, with different patterns when compared to R-modafinil (10 mg/kg i.p.); in the nucleus accumbens shell, a low and transitory increase of dopamine was observed only after (S)-CE-123. Neither (S)-CE-123 nor R-modafinil initiated the emission of 50-kHz ultrasonic vocalizations, a behavioral marker of positive affect and drug-mediated reward. Our data support previous reports of the procognitive effects of (S)-CE-123, and show a minor impact on reward-related dopaminergic areas.Treatments for cognitive impairments associated with neuropsychiatric disorders, such as attention deficit hyperactivity disorder or narcolepsy, aim at modulating extracellular dopamine levels in the brain. CE-123 (5-((benzhydrylsulfinyl)methyl) thiazole) is a novel modafinil analog with improved specificity and efficacy for dopamine transporter inhibition that improves cognitive and motivational processes in experimental animals. We studied the neuropharmacological and behavioral effects of the  The mesocorticolimbic dopamine system is involved in processing motivation, salience, and reward, with dopamine also being a neuromodulator in brain areas that are involved in diverse cognitive processes. Reuptake and compartmentalization have a pivotal role in neurotransmission, balancing extracellular/cytosolic synaptic neurotransmitters. The dopamine transporter (DAT) is located in the presynaptic plasma membrane, where it spatially and temporally produces an accumulation of dopamine . Given iDespite the positive effects of drugs improving cognitive functions, side effects  have to S enantiomer of CE-123, (S)-CE-123, which was shown to be more potent at inhibiting DAT than the R enantiomer , of time , a significant treatment × time interaction , dose × time interaction , and treatment × dose × time interaction . Tukey’s post hoc tests showed a significant increase of dialysate dopamine at 40 min after the administration of (S)-CE-123 (10 mg/kg i.p.), as compared to basal value, to vehicle and to the lower dose of (S)-CE-123 (1 mg/kg i.p.). A Tukey’s post hoc tests also showed a significant increase of dopamine at 100, 120, and 160 min after administration of the higher dose of R-modafinil (10 mg/kg i.p.) with respect to basal value, from 100 to 160 min concerning the vehicle group, but also at 80, 100, 120, and 160 min with respect to the lower dose of R-modafinil (1 mg/kg i.p.). Moreover, Tukey post hoc tests revealed significant differences at 160 and 180 min after the administration of 5 mg/kg i.p. of R-modafinil with respect to both basal value and vehicle-treated animals, and significant differences at 160 min between animals treated with (S)-CE-123 (10 mg/kg i.p.) or R-modafinil (10 mg/kg i.p.). Considering IL/PrL cortex, a three-way ANOVA showed a main effect of dose , and a significant dose × time interaction . Tukey’s post hoc tests showed a significant increase of dialysate dopamine at 40 min after the administration of (S)-CE-123 (10 mg/kg i.p.), to the basal value; it also showed significant differences at 40 min between rats treated with (S)-CE-123 (10 mg/kg i.p.) and R-modafinil (10 mg/kg i.p.).Considering the NAc shell, three-way ANOVA showed a main effect of time [FS)-CE-123 and R-modafinil on the emission of 50-kHz USVs, a behavioral marker of positive affect and drug-induced reward, in a subset of freely moving rats during brain microdialysis. Acute administration of (S)-CE-123  did not increase the numbers of 50-kHz USVs emitted, as compared to vehicle administration  = 0.76, p = 0.54) and no significant treatment × time interaction  = 0.77, p = 0.76), although a significant effect of time was observed  = 4.92, p = 0.00002). Similarly, acute administration of R-modafinil  did not elevate the numbers of 50-kHz USVs emitted, as compared to vehicle administration  = 0.79, p = 0.51) and time  = 1.06, p = 0.39), and no significant treatment × time interaction  = 1.28, p = 0.18). We tested the effect of 1, 5, and 10 mg/kg  in anesthetized rats, while (S)-CE-123 produced a dose-dependent decrease. Dopaminergic neuromodulation in frontal lobes and in subcortical brain areas is involved in processing cognitive skills, such as attention and associative learning ,44, and R-modafinil (5 and 10 mg/kg i.p.) increased extracellular dopamine levels with a delayed and protracted effect, while (S)-CE-123 (10 mg/kg) rapidly stimulated dopamine transmission. This is consistent with a microdialysis study showing that 128 mg/kg modafinil enhanced dopamine levels in prefrontal cortex, with the highest dopamine levels occurring within 2–3 h [in 2–3 h . S)-CE-123 and R-modafinil for neuropharmacological effects in rat brain areas related with reinforcement. The selective increase of dopamine neurotransmission in the NAc shell plays a pivotal role in processing reward and motivated behaviors following intake of drugs of abuse [S)-CE-123 does not increase dopamine levels (except for a low and transitory increase at 40 min at the 10 mg/kg dose). In contrast, at 10 mg/kg a downward trend of dopamine levels was observed from 80 to 180 min following administration. This lack of stimulation of dopamine transmission in the NAc shell is consistent with what observed at the dose of 24 mg/kg ip [S)-CE-123 at doses of 10 and 100 mg/kg increased dopamine extracellular levels in the NAc shell of mice [S)-CE-123 in the metabolic pathway of species (mice vs. rats), together with the specificity of the brain area implanted, might be the reason of the lack of increase observed in the present study. Similar to (S)-CE-123, none of the tested doses of R-modafinil stimulated dopamine transmission in the NAc shell. This observation is inconsistent with previous findings, since both Mereu et al. (2017) [R-modafinil.It is well established that cognitive and executive functions are modulated by dopamine, depending on cellular localization of postsynaptic receptors and their downstream signaling, which can be excitatory or inhibitory ,50. D1- of abuse ,55. Our mg/kg ip , althoug of mice . Differe. (2017)  and Keig. (2017)  reported. (2017) , prelimi50 6.4–13 μM) and NET activity (IC50 35.6–182 μM), albeit with lower potency in the case of the latter [50 = 4.6 μM) without acting as a substrate [Differences between the mechanisms of action of (S)-CE-123 and the parent compound modafinil might explain our results. Multiple neurotransmitter systems have been implicated in modafinil activity ,58, as se latter ,60,61. Oubstrate . Moreoveubstrate .S)-CE-123 or R-modafinil up to the 10 mg/kg cumulative dose. Analogous doses of well-known drugs of abuse targeting DAT (such as cocaine and amphetamine) have been consistently shown to reduce dopamine neuron firing [Our in vivo electrophysiological recordings showed that VTA dopamine cell firing frequency and bursting activity were not substantially affected by the intravenous administration of either -CE-123 and R-modafinil. Indeed, the activation of dopamine receptors in the NAc shell has been shown to be a critical mechanism that initiates the emission of 50-kHz USVs [S)-CE-123 and R-modafinil  did not alter 50-kHz USV emissions in adult male rats, consistent with the weak effects that both drugs elicited on dopamine release in the NAc shell. The lack of effect of (S)-CE-123 on 50-kHz USV emissions marks a clear difference with other DAT-inhibiting compounds with abuse potential.As a behavioral marker of positive affect and drug-induced reward in rats , we evalkHz USVs ,70. MorekHz USVs ,74,75,76S)-CE-123 and R-modafinil in the mesocorticolimbic system are in accordance with published studies showing the behavioral and neurochemical peculiarities of analogue compounds, despite their chemical similarities to the drug from which they were derived [S)-CE-123 are effective in frontal brain areas associated with cognition, with minimal effect in brain areas typically related to the rewarding properties of drugs of abuse.In conclusion, the differential effects of ( derived . The pre


Anopheles gambiae complex is comprised of eight morphologically indistinguishable species and has emerged as a model system for the study of speciation genetics due to the rapid radiation of its member species over the past two million years. Male hybrids between most An. gambiae complex species pairs are sterile, and some genotype combinations in hybrid males cause inviability. We investigated the genetic basis of hybrid male inviability and sterility between An. coluzzii and An. quadriannulatus by measuring segregation distortion and performing a QTL analysis of sterility in a backcross population. Hybrid males were inviable if they inherited the An. coluzzii X chromosome and were homozygous at one or more loci in 18.9 Mb region of chromosome 3. The An. coluzzii X chromosome has a disproportionately large effect on hybrid sterility when introgressed into an An. quadriannulatus genetic background. Additionally, an epistatic interaction between the An. coluzzii X and a 1.12 Mb, pericentric region of the An. quadriannulatus 3L chromosome arm has a statistically significant contribution to the hybrid sterility phenotype. This same epistatic interaction occurs when the An. coluzzii X is introgressed into the genetic background of An. arabiensis, the sister species of An. quadriannulatus, suggesting that this may represent one of the first Dobzhansky–Muller incompatibilities to evolve early in the radiation of the Anopheles gambiae species complex. We describe the additive effects of each sterility QTL, epistatic interactions between them, and genes within QTL with protein functions related to mating behavior, reproduction, spermatogenesis, and microtubule morphogenesis, whose divergence may contribute to post-zygotic reproductive isolation between An. coluzzii and An. quadriannulatus.The  The study of speciation genetics and genomics has largely focused on the evolution of hybrid dysfunction  because Drosophila (Solanum sibling species pairs (Post-zygotic isolation occurs as Dobzhansky-Müller incompatibilities (DMI) arise between populations evolving in allopatry . Upon seosophila  and Solaes pairs .Mimulus guttatus, OdsH gene in Drosophila simulans × D. mauritiania backcross males, JYAlpha gene in D. melanogaster × D. simulans backcross males, Arabidopsis thaliana strains, cis- and/or trans-acting factors  or mutation-order speciation scenarios  . These m factors , and seg factors .Anopheles (An.) gambiae complex is comprised of eight morphologically indistinguishable species and has emerged as a model system for the study of speciation genetics due to the rapid radiation of its member species over the past two million years  (An. quadriannulatus A) (The on years . All memoluzzii)  to almoslatus A) . From a latus A)  because Anopheles gambiae complex are fertile, but, with the exception of the An. gambiae – An. coluzzii species pair, males range from semi- to completely sterile , and those that have the standard arrangement, including An. quadriannulatus.F1 hybrid female progeny of crosses between species of the  sterile . A previce versa , 2005. ODrosophila  F1 × An. quadriannulatus backcross, and identified regions of the An. coluzzii genome that contribute to hybrid male sterility and inviability when introgressed into an An. quadriannulatus genetic background. We discuss how these QTL relate to those identified in the (An. coluzzii × An. arabiensis) F1 × An. arabiensis backcross  hybrids were reared and backcrossed to An. quadriannulatus males en masse. Consistent with crosses between An. coluzzii and An. arabienesis, the Xag inversion was expected to suppress recombination between the X chromosomes of An. coluzzii and An. quadriannulatus.We performed a backcross between the SUA2La strain of 2La inversion is also fixed between the mosquito strains used in this cross, limiting our mapping resolution in this region of the genome. While the 2La is polymorphic within An. coluzzii, and An. coluzzii strains with the standard arrangement (co-linear with An. quadriannulatus) are available, we used the same An. coluzzii strain as The The testes of male hybrids were dissected and squashed between a microscope slide and a cover slip. Each hybrid male mosquito was assigned a sterility phenotype following DNA extractions were performed on a Qiagen Biosprint DNA extraction machine . Extracted DNA was suspended in 200 μl Qiagen elution buffer and stored at −20°C. Each mosquito was genotyped using the restriction enzyme-based, genotype-by-sequencing approach according to the multiplexed shotgun genotyping (MSG) approach published by MseI, which cuts the An. gambiae genome on average every 183.2 bp. Samples were digested for 3 h at 37°C, followed by a 20 min, 65°C enzyme deactivation step. Non-ligated bar-coded adapters were removed using an isopropanol precipitation, and bar-coded samples with unique adapters were pooled (96 per plate). Pooled samples were precipitated via centrifugation and re-suspended in TE buffer. Next, DNA was bead-purified using an Agencourt AMPure PCR purification kit. Bar-coded, purified DNA was size selected to a range of 250–400 bp using a Pippen Prep and then amplified using a Phusion High-Fidelity PCR kit. Samples were amplified using a common primer, and an indexed, library-specific primer (one per plate) that also incorporates the Illumina flow-cell adapter. Thus, individuals were identifiable to a library prep by their index and to the individual level by their barcoded adapter. Library amplification (PCR) was performed at a range of 12–18 cycles to identify the minimum number necessary to amplify the library to a target of 10–30 ng/μL (post clean up) in an effort to mitigate the incorporation of PCR errors. Amplified libraries were bead-purified using an Agencourt AMPure PCR purification kit. Library size distribution, integrity, and DNA concentration was confirmed by running each library on a NanoDrop and BioAnalyzer prior to sequencing. We phenotyped and sequenced a total of 841 male backcross hybrids. Hybrids were sequenced at the Texas A&M Genomics and Bioinformatics core facility on an Illumina HiSeq 2500 using 125 bp, single-end chemistry to 0.5–2.0x depth per individual.Briefly, 10 ng of genomic DNA of each mosquito was digested with the restriction enzyme An. coluzzii females and ten An. quadriannulatus females in order to identify fixed genetic difference between parental strains that would be diagnostic of the origin of genomic regions introgressed from An. coluzzii into An. quadriannulatus in the backcross. Libraries were sequenced on a single lane of Illumina HiSeq 2500 using 150 bp, paired-end chemistry to an average depth of 110.5x (An. coluzzii) and 66.9x (An. quadriannulatus). Additionally, we prepared and sequenced MSG libraries for 192 An. coluzzii and 192 An. quadriannulatus females to further validate ancestry informative markers and tune the MSG software error model.We also performed pool-seq of ten An. coluzzii pool-seq reads were aligned to the An. gambiae AgamP4.4 reference genome using the BWA-MEM alignment algorithm (An. coluzzii was created by updating the AgamP4.4 reference genome (vectorbase.org) with SNPs that had read depth between greater than 10 and less than 1000, map quality greater than 20, and a phred-scaled quality score for the alternative allele greater than 26. The An. quadriannulatus pseudo-reference was created in a similar fashion, but by aligning it to the An. coluzzii pseudo-reference and updating this with fixed SNPs (An. coluzzii – An. quadriannulatus divergent) that met the thresholds above.lgorithm  and SNPslgorithm . SNPs wiWe used Trimmomatic version 0.30  to trim 1. We considered a marker to be informative if its conditional probability of being homozygous differed by more than 0.10 from its neighboring marker. Missing genotypes were imputed using the hidden Markov model implemented in the sim.geno function of R/qtl  homozygous An. quadriannulatus autosomal loci that interact epistatically with the An. coluzzii X in individuals that are otherwise primarily heterozygous at autosomal loci (recovering viability despite the inheritance of a non-recombinant An. coluzzii X chromosome). Thus, we also split the dataset into individuals that had inherited the An. quadriannulatus X (N = 568) and An. coluzzii X (N = 211), respectively, and performed CIM (as above) on these datasets separately.Ultimately, we took three approaches to identify sterility QTL in the dataset. The first was to scan for QTL using the composite interval mapping and calculating the logarithm of these odds (LOD) score every 1 cM. When calculating LOD scores we controlled for the genetic background of adjacent markers by using 15 marker covariates across the genome. Due to the skew of the phenotype scores toward “normal,” and severe segregation distortion of the X chromosome, we hypothesized that the QTL identified through CIM of the full dataset were in fact comprised of two types: (1) autosomal An. quadriannulatus X or An. coluzzii X datasets, respectively, by adding each peak independently to the multiple imputation QTL model that also contained the highly significant X chromosome QTL. After identifying all peaks that had a significant additive effect in the multiple imputation QTL model, we tested epistatic interactions between each, in a pairwise fashion, by adding each of these interactions to the model independently. This approach allowed us to identify peaks (and their interactions) which, despite having non-significant LOD scores in the CIM permutation test, were found to have a significant impact on sterility when incorporated into the multiple imputation QTL model.Next, we tested all peaks identified by CIM, regardless of significance or LOD score, in the full dataset, and any peaks that were unique to either the vectorbase.org) genome browser using the “Export Data” function. Next we used the PANTHER classification system (pantherdb.org) to search for the protein annotations and functions of these genes. Finally, we performed a protein network analysis on the entire gene list using STRING  from expected homozygosity.We identified regions of the An. coluzzii pool-seq data, we generated the An. coluzzii pseuo-reference by incorporating 4,391,200 filtered SNPs into the An. gambiae s.s. AgamP4 reference genome. To construct the An. quadriannulatus pseuo-reference, we updated the An. coluzzii pseudo-reference with 4,242,426 An. quadriannulatus SNPs that were found to be fixed between the An. quadriannulatus and An. coluzzii pool-seq samples. MSG data is inherently low coverage, so not all fixed differences between the pool-seq datasets were subsequently used for hybrid genotyping.After whole genome alignment, SNP calling, and variant filtering of the An. coluzzii × An. quadriannulatus backcross hybrid males. Ultimately, we trimmed the dataset to 860 non-redundant markers . These markers were used to calculate a genetic map  inherited a non-recombinant An. quadriannulatus X chromosome, 26.1% (N = 211) inherited a non-recombinant An. coluzzii X, and 3.5% (N = 29) a recombinant X. The mean sterility score  was 2.62 (SD = 2.34). The majority of backcross mosquitoes  were fully fertile (sterility score = 1).The genetic length of the X chromosome was expected to be low due to the presence of the An. coluzzii X and An. quadriannulatus X datasets, respectively, be comparing the proportion of An. quadriannulatus homozygosity at each locus to the genome-wide average in the full dataset. It is important to note that this analysis was performed on a per locus basis, and is agnostic of interactions between loci.Segregation distortion was previously documented in male and female hybrids between member species of this complex . FurtherAn. quadriannulatus X dataset, 67.3% (N = 296) of chromosome 2 loci and 18.4% (N = 72) of chromosome 3 loci showed significant segregation distortion  of chromosome 2 loci and 69.6% (N = 273) of chromosome 3 loci showed significant segregation distortion. Interestingly, 40.8% of chromosome 3 loci (spanning a 18.9 Mb region) lack homozygotes in all genotyped individuals, indicating individuals that inherited the An. coluzzii X and were An. quadriannulatus homozygous at loci inside this region were completely inviable  was found in the full and both X chromosome split datasets, suggesting that it contains loci involved in autosomal-autosomal incompatibilities   . None ofis cross .An. quadriannulatus X dataset, but not the An. coluzzii X dataset, indicating that An. coluzzii introgressions into a predominantly An. quadriannulatus genetic background are driving this signal. QTL “3L-A” in the An. coluzzii × An. quadriannulatus dataset overlaps with a QTL in the An. coluzzii × An. arabiensis dataset  of homozygous genotypes at the 3L-A and 3L-B QTL differs significantly from expectation   and 67.46 cM (QTL “3L-B”) . These a dataset . The 3L-luzzii X , and eacing HWE) . Thus, tAn. coluzzii X chromosome have on average higher sterility scores because in these backcrosses there are more opportunities for An. quadriannulatus autosomal – An. coluzzii X incompatibilities than vice versa  dataset. The QTL discussed above, and those reported in ce versa .F-test p < 0.01) (F-test p = 5.60E-06). In both models, additive effects of autosomal loci, and the epistatic interaction between the X and 2L QTL, each explained less than 1% of the variance in hybrid sterility. The “epistasis allowed” model explained a higher proportion of the variance and had a higher model LOD (225.78) when compared to the “additive only” model (221.25)  . After t(221.25) .An. quadriannulatus homozygous (QQ) vs. An. quadriannulatus/An. coluzzii heterozygous (QC)] at autosomal QTL and the X chromosome  dominant An. coluzzii sterility gene that causes incompatibilities with the An. quadriannulatus background.To better understand the direction of the effect of each QTL in the full dataset, we compared the phenotypes of each individual given their genotype [romosome . Each auAn. coluzzii × An. arabiensis cross  parent X chromosome  of each gene using PANTHER. Due to the lack of resolution on the X chromosome, we excluded this chromosome from this analysis. We identified 349 genes within all autosomal QTL  the cumulative effect of the X chromosome accounted for greater than 60% of the phenotypic variation. These results support the theory of large X effect on hybrid male sterility due to its hemizygosity in the heterogametic sex  where recessive sterility genes are expressed, but cannot discount the role of faster X evolution, which we did not test. The large proportion of variance explained by the X in our An. coluzzii × An. quadriannulatus backcross contrasts with the An. coluzzii × An. arabiensis backcross (to An. arabiensis) performed by An. coluzzii × An. arabiensis backcross resulted, at least in part, from the inviability observed when hybrids inherited the An. coluzzii X  have experimentally determined, direct interactions with each other, while another interaction is predicted due to co-expression (AGAP004724 and AGAP010790)  failure associated with sperm individualization and maturation  within autosomal P010790) . While tre sperm . Infertituration . Recent e testes .An. coluzzii × An. quadriannulatus cross. Interestingly, another tubulin-specific chaperone, E-like, is required for sperm individualization and male fertility in Drosophila (Sperm flagellum elongate from the spermatid head after meiosis. The axoneme is the central component of the sperm flagellum and has a 9 + 2 structure of a central pair of microtubules surrounded by nine doublet microtubules. Doublet microtubules have an inner and outer dynein arm that serve to attach and detach to neighboring doublet microtubules, and function in the motor activity of the flagellum. The axoneme is surrounded by a mitochondrial sheath that serves to provide ATP and power motor activity. Tektins are located near the junction points of dynein arms and neighboring microtubules . Seven oosophila .Drosophila (Drosophila lingerer mutant males fail to withdraw their genitalia after copulation (An. gambiae complex may yield insight into the specific alleles responsible for hybrid male sterility between An. coluzzii, An. quadriannulatus, and An. arabiensis.The three genes without tubulin/microtubule functions are: transcription factor ken, protein lingerer, and a checkpoint protein Hus1-like. Mutations in the ken and barbie transcription factor result in malformed genitalia in osophila . lingerepulation . Finallypulation . Future An. coluzzii and An. quadriannulatus. We have also narrowly defined autosomal QTL which have a statistically significant contribution to sterility despite their smaller effect sizes in comparison to the X. The shared, autosomal sterility QTL between the An. coluzzii × An. quadriannulatus and An. coluzzii × An. arabiensis backcrosses suggest that these may represent important autosomal regions involved in the post-zygotic reproductive isolation of An. coluzzii from its sister species that diverged early in the radiation of the An. gambiae species complex. Future analyses into factors contributing to female hybrid sterility in the An. coluzzii × An. quadriannulatus cross will add to our knowledge of the genetics of speciation in this evolutionarily and medically important group of human malaria mosquitoes.In this study we have determined that the X chromosome accounts for that majority of the variance in the hybrid male sterility phenotype in a cross between https://www.ncbi.nlm.nih.gov/bioproject/PRJNA647477 Genotype and phenotype data can be found on Dryad here: https://doi.org/10.5061/dryad.bvq83bk6d, with a a direct download of the dataset available here: https://datadryad.org/stash/share/kw5d5Pio8DMRJuogJtE5x1kf5zC81ViDnH9FNKOAxxs.Raw genome sequencing data can be found on the NCBI SRA here: KD, WT, and MS designed the experiment. KD and MS performed the experiment and wrote the manuscript. KD analyzed the data. WT edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Eco-cognitive computationalism sees computation in context, exploiting the ideas developed in those projects that have originated the recent views on embodied, situated, and distributed cognition. Turing’s original intellectual perspective has already clearly depicted the evolutionary emergence in humans of information, meaning, and of the first rudimentary forms of cognition, as the result of a complex interplay and simultaneous coevolution, in time, of the states of brain/mind, body, and external environment. This cognitive process played a fundamental heuristic role in Turing’s invention of the universal logical computing machine. It is by extending this eco-cognitive perspective that we can see that the recent emphasis on the simplification of cognitive and motor tasks generated in organic agents by morphological aspects implies the construction of appropriate “mimetic bodies”, able to render the accompanied computation simpler, according to a general appeal to the “simplexity” of animal embodied cognition. I hope it will become clear that eco-cognitive computationalism does not aim at furnishing a final and stable definition of the concept of computation, such as a textbook or a different epistemological approach could provide: I intend to take into account the historical and dynamical character of the concept, to propose an intellectual framework that depicts how we can understand not only the change of its meaning, but also the “emergence” of new forms of computations. Eco-Cognitive Computationalism sees computation in context, exploiting the ideas developed in those projects that have originated the recent views on embodied, situated, and distributed cognition: computation is seen as active in physical entities appropriately transformed so that they become what I call cognitive mediators, in which data can be encoded and decoded to obtain productive results. This eco-cognitive approach to the concept of computation is strictly related to the tradition of research concerning the so-called distributed and embodied cognitive systems. The “distributed” approach sees cognition as a socially distributed process, pragmatically oriented, and affirms that cognitive processes can be better analyzed as situated in and distributed across material artifactual circumstances, in which the “ecological” view also emphasizes the role of the agent-environment interaction. The theory of distributed cognition was created by Edwin Hutchins  the possession of a human cortex (say) would be virtually useless if no attempt was made to organize it. Thus if a wolf by a mutation acquired a human cortex there is little reason to believe that he would have any selective advantage”  has to be already available, but also, as I have noted above, together with a small community that can grant the passage of information across generations.When an unorganized machine (such as an infant cortex) is submitted to suitable interferences, its behavior changes and step after step the machine becomes organized . Not only is the existence of a human cortex evolutionarily justifiable only in terms of its organization in the framework of a antage” ( p. 16). discipline and initiative, which Turing considers the two main aspects of a process that has to be studied as it happens in humans to “copy it in machines” .educated to produce certain kinds of modification. In this last case, education coincides with programming, which is an imitation of the human case: we can modify machines thanks to programming to the aim of reaching some good and interesting responses. The concept of  Logical Computing Machine (LCM) emerges in this epistemological atmosphere and, on the basis of this abstract theoretical tool, also one of the  Practical Computing Machines (PCM). These are digital computers  that can be built and “educated”, able to imitate the behavior of a human computer very well. I have already anticipated that, to show Turing’s externalization of both the abstract  and the concrete  entities, I have called them mimetic minds  with nonlinear activation functions and with recurrent connections that have a random but bounded strength; this is referred to as a dynamic reservoir. These neurons are randomly connected to input streams, and the dynamics of the input is then spread around and transformed in the reservoir, where it resonates (or “echoes”—hence the term “echo-state networks”) for some time. It turns out that tapping into the reservoir with simple output connections is often sufficient to obtain complex mappings of input stream to output stream that can approximate the input–output behavior of highly complex nonlinear dynamical systems. During training, the weights from the input streams and between the reservoir neurons are left intact; only the output weights—from the reservoir to the output layer-are modified by a learning algorithm . The complexity of the training task has been greatly reduced  by exploiting the reservoir to perform a spatiotemporal transformation of the input stream . Furthermore, if feedback loops from the output back to the reservoir are introduced and subject to training, the network can be trained to generate desired output streams autonomously.In the first case, an example is the passive dynamic walker ; in the second case, the classical case is illustrated by the Gecko feet, which can in turn be considered as active extensions of the passive walker, thanks to actuators and sensors ; also the case of the eye of the fly, which not only concerns movement, but also cognitive abilities related to perception, is another good example; the third case refers to the so-called Reservoir Computing, abstractly proposed by the neural network community, and optimally extended to the role of physical devices that can operate as a reservoir ), in which the body is effectively domesticated for computation. Reservoir computing is not only limited to morphological computations, but, as I have just said, originates from research on neural networks, and contemplates a collection of neurons, as Müller and Hoffmann summarize,The amazing novelty is that some physical entities can work as a reservoir as well, giving origin to the so-called Physical RC, which can also be the body of an agent: indeed, biological bodies interacting with their environments can present the needed properties—high dimensionality, nonlinearity and fading memory  onto its high-dimensional state space in a nonlinear fashion. In that sense the reservoir takes the role of a kernel . In addition, the reservoir, being a dynamical system, has the inherent property to integrate input information over time, which is obviously beneficial for any computation that needs information on the history of its input values. It is important to note that the reservoir is not altered during the learning process. Although it is randomly initialized, its dynamic parameters are fixed afterwards. In order to learn to emulate a desired input output behavior , one has to add a linear output layer, which simply calculates a linearly weighted sum of the signals of the high-dimensional state space of the reservoir. These output weights are the only parameters that are adapted during the learning process.At the core of RC lays the so-called To further examine the ways of domesticating—beyond the usual world-wide widespread use of silicon devices—non-stardard entities for computation, we need describe some cognitive and epistemological aspects of the so-called RC (Reservoir Computing). In this case, a classical “digital” computational controller tool —that already needs its own “conventional” physical embodiment—is enhanced by the fact that some cognitive “virtues” are outsourced to the morphology of physical entities with the aim of getting the good fruits of an “analog” computation. Where should we draw the line between morphological computation and pure digital computation? Hauser et al. Implementation of the so called Physical RC involves real physical bodies that are used as reservoirs and as computational sources. Obviously, both the entities involved in classical highly engineered silicon devices and in Physical RC exploitation of bodies do not “know” that they are part of a computational device. They simply conform to the laws of physics and they respond spontaneously. In the case of Physical RC, we have to note “[…] that this also implies that the body does not over—nor under—compensate, since it is a stable physical system. The proposed setup simply adds some linear readouts to the body to complete the computation. The body would react exactly the same, if there were no readouts at all. If this output is used, e.g., in a feedback loop as a control signal for the robot, the behavior of the robot of course should be different if we close the loop by adding the readout” Hence, the body is so to speak “ignorant” digital processing and, from the point of view of their macroscopic features, are stable, unperturbable, and robust with respect to the external influences different from the processes of encoding and decoding  of the physical adopted reservoir a kind of morphosis   as silicon devices or robots endowed with an abstract controller  don’t have any deliberate computation in the physical layer, hence, any computation in the robot is carried out only in the abstract controller layer” osture) mimetic bodies, that is, bodies that are able to mime various cognitive mediating tasks. I call them mimetic exactly in Turing sense: Turing says by “mimicking education, we should hope to modify the machine until it could be relied on to produce definite reactions to certain commands” . mmands”  compute the shortest path through a leaf in photosynthesis? Is the human mind a computer? A dog catching a stick? A stone sitting on the floor? One answer is that they all are – that everything that physically exists is performing computation by virtue of its existence. Unfortunately, by thus defining the universe and everything in it as a computer, the notion of physical computation becomes empty. To state that every physical process is a computation is simply to redefine what is meant by a “physical process”—there is, then, no non-trivial content to the assertion. A statement such as “everything is computation” is either false, or it is trivial; either way, it is not useful in determining properties of physical systems in practice.The problems of pancomputationalism is nicely summarized in the following passage by Horsman et al.:The assumption of pancomputationalism clearly states that everything is computational . This vicognitive niches, hugely endowed with informational  processes  enhanced phonological storage may have freed language from the laconic and its confinement to present tense and simple imperatives to rapidly-spoken speech and the use of future tense—the linking of past, present, and future, and the use of the subjunctive […]. Although real enemy’s actions might be anticipated, imaginary enemies could be envisioned and other intangible terrors could be given life. Great anxieties could arise with novel vistas ” , which ensure the stability of a concept, and the physical and material processes that ensure the stability of the actual object represented by the concept. A discretization of knowledge that—long before the computational turn—did not have a marginal role in the recent history of human cognition. The internalization of phonemes  has been etc.)” .Notwithstanding the triumph of discretization, in western written natural languages but also in philosophical and logical knowledge, from Democritus to Descartes and from the modern XIX century axiomatics to the computational turn, we are still facing the conundrum represented by the fact that, however, this simple reality of small components is actually very complex, as recent scientific research into the dynamical systems theory, quantum physics, and biology demonstrates. For example, it is difficult to study the cell only by referring to its constituents, and also its “wholeness” is fundamental. The suspect is that, when we use computational devices, which are discrete machines, to produce knowledge about physical and biological systems, some serious expressive limits arise: it is unlikely that these machines can play the role of instruments for directly building scientific intelligibility regarding complex objects/systems such as physical and biological entities   stronglimitation, as developed by digital machines and their “modeling capacity”, and the idea of scientific knowledge as generation of rational intelligibility is a core problem of the dynamical approach [imitation capacity of machines and the modeling power of mathematics: the double pendulum, which is a perfect deterministic machine, only expressed by two equations, is sensible to minor variations, below the threshold of observability: it is a typical chaotic deterministic system, and it is extremely difficult to represent it by a mimetic machine [The deep difference between the idea of scientific knowledge as simple approach . As I al machine . Longo aresembles or imitates causality, always realizing Laplacian and predictable processes. The explanation of this conundrum resorts to the fact that, at the roots of digital data, there is a discrete topology, but physical measurement is always an interval that is optimally represented by continuous mathematics. Even if sometimes the use of mathematical equations in physics does not have a predictive power, this provides a knowledge characterized by a rich qualitative epistemological value, able to make intelligible the physical causality of the system [Only “mathematical” models can explain the structure of physical causality of the a system at play, and digital simulation only e system . Finallye system  Longo coIn summary, digital simulation—even if epistemological useful at the level of intermediate modeling during the processes of scientific research and discovery, as I have already indicated above in this section—produces an epistemologically distorting result due to its simply mimetic quality.In this article, taking advantage of an approach I called “eco-cognitive computationalism”, I have illustrated Turing’s original intellectual perspective that furnished the conceptual framework able to show how, thanks to an imitation of the the evolutionary emergence in humans of information, meaning, and of the first rudimentary forms of cognition, the subsequent invention of the Universal Practical Computing Machine is achieved, that computer that, from the perspective offered by Turing, I call “mimetic mind”. In the second part of the article, I have exploited this framework to illustrate the recent results of morphological computation in which the emphasis on the simplification of cognitive and motor tasks has rendered possible the construction of appropriate “mimetic bodies“ able to render accompanied computations simpler, according to a general appeal to the “simplexity” of animal embodied cognition. I have stressed that, in the case of morphological computation, we can surely conclude that we are facing a new activity of what we can call “distributed computation”: the promise of morphological computation principles in robot design can originate a new generation of robots with better adaptability and restricted number of required control parameters. Finally, I have also dedicated a short discussion to the concepts of paninformationalism and pancomputationalism, showing that the framework of distributed computation helps us see them in a more naturalized and prudent perspective, avoiding ontological or metaphysical considerations. The last section of the article is devoted to illustrating the related problems regarding the epistemological limitations of computation modeling when used to simulate the behavior of a physical or a biological system .


SAMDH1 gene may alter the enzymatic expression and influence the course of infection by the virus. The present study investigated the effect of polymorphisms on HTLV-1 infection susceptibility and on progression to disease in 108 individuals infected by HTLV-1 (47 symptomatic and 61 asymptomatic) and 100 individuals in a control group. SAMHD1 rs6029941 (G/A) genotyping and HTLV-1 proviral load measurements were performed using real-time PCR and plasma IFN-α was measured by ELISA. Polymorphism frequency was not associated with HTLV-1 infection susceptibility or with the presence of symptoms. The proviral load was significantly higher in symptomatic individuals with the G allele (p = 0.0143), which presented lower levels of IFN-α (p = 0.0383). SAMHD1 polymorphism is associated with increased proviral load and reduced levels of IFN-α in symptomatic patients, and may be a factor that contributes to the appearance of disease symptoms.SAMHD1, a host dNTPase, acts as a retroviral restriction factor by degrading the pool of nucleotides available for the initial reverse transcription of retroviruses, including HTLV-1. Polymorphisms in the  HTLV-1 is responsible for the development of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T-cell leukemia/lymphoma (ATLL) and is associated with other inflammatory syndromes, such as rheumatoid arthritis, dermatitis, and uveitis, in addition to autoimmune diseases  that acts as an intrinsic factor of retroviral restriction, degrading the pool of nucleotides available for the initial reverse transcription, limiting the replication of retroviruses, including HTLV-1 , seems to alter enzyme expression, where the A allele is associated with higher levels of SAMHD1 expression and the G polymorphic allele is associated with lower levels  on the proviral load and the development of symptoms of HTLV-1-associated diseases.Genetic variations in the Chlamydia trachomatis or syphilis viruses, to compare polymorphism frequencies.The present study included blood samples from 108 individuals infected with HTLV-1  treated at the Tropical Medicine Center outpatient clinic of the Federal University of Pará. The patients were of both sexes, were older than 18 years of age and had not been treated with glucocorticoids. The control group included 100 individuals at risk of infection but not infected with the HTLV-1/2, HIV-1, hepatitis B or C, A 10 mL blood sample was collected by intravenous puncture using a vacutainer system containing ethylenediaminetetraacetic acid as an anticoagulant. The samples were centrifuged and separated into plasma and a leukocyte mass. The leukocyte samples were used to extract genomic DNA for analysis of the SAMHD1 rs6029941 (A/G) polymorphism and quantification of the proviral load.DNA was extracted from peripheral blood leukocytes using the Puregene kit  according to the manufacturer's protocol, which included cell lysis, protein precipitation, and DNA precipitation and rehydration. DNA was quantified using a Qubit® 2.0 fluorometer  and Qubit™ DNA assay kit reagents , following the protocol recommended by the manufacturer.Proviral load was quantified using a quantitative real-time PCR using three target sequences, synthesized through the TaqMan® system , according to a previously described protocol  containing primers and specific TaqMan® probes for amplification of the target sequence . The reaction contained 1× MasterMix, HPlasma IFN-α was measured by the enzyme-linked immunosorbent assay (ELISA) Invitrogen Human IFN alpha ELISA Kit , which uses specific monoclonal antibodies to detect the cytokine and followed the manufacturer's instructions.n” and %).The genotype frequencies were estimated by direct count. The allele frequency was calculated using the formula: F = 2 × number of homozygous individuals + number of heterozygous individuals/total number of individuals. The sum of the two alleles must equal 1. This is the standard form of scientific literature in the field of genetics to describe allele frequencies  test. The proviral load and plasma IFN-α were compared between groups using the non-parametric Kruskal-Wallis and Mann-Whitney test. All tests were performed using BioEstat 5.3 software. Associations with p < 0.05 were considered statistically significant.Differences between genotype frequencies observed in the investigated groups were calculated by the χSAMDH1*G) in individuals with the virus, but without statistical significance  polymorphism were similar between individuals infected with HTLV-1 and the control group, with a higher frequency of the polymorphic allele . In contrast, median IFN-α levels were lower in individuals with polymorphic genotypes  but without statistical significance (p = 0.1246).The median of proviral load was higher in individuals infected carrying the polymorphic allele , and significant difference was observed between wild-type and polymorphic genotypes, AG and GG (*G) in homo and heterozygosis (AG and GG), which are associated with reduced expression of the restriction factor. The viral load was significantly higher in symptomatic individuals with polymorphic genotypes, p = 0.0143 , related to greater expression of SAMHD1, compared to individuals with genotypes expressing the polymorphic allele  polymorphism was not associated with infection susceptibility or with the presence of HTLV-1-related symptoms. These results may be related to the small sample size used in the study. Although the Amazon region is endemic for HTLV-2 infection, found mainly in the indigenous population  polymorphism with the symptoms of the diseases is that it may not be associated with the development of symptoms of all types of diseases associated with HTLV-1, because, although they are of inflammatory etiology, they activate different immunological mechanisms  polymorphism and variations in the HTLV-1 proviral load. The GG polymorphic genotype, related to lower enzyme levels  polymorphism may influence HTLV-1 infection in the evaluated tri-hybrid population. However, because this is the first study that investigated the association of polymorphism in HTLV-1 infection, it will also be necessary to analyze its relationship in other different infected ethnic groups to determine its relevance in other populations.An important aspect that needs to be considered is the genetic background of the population analyzed in this study, which results from interethnic crossbreeding of Europeans, Indians, and Africans  was based on its influence on changes in gene expression and because it has not yet been evaluated for HIV and HTLV infection. Although the frequency of polymorphism is not associated with the presence of disease symptoms, it was associated with a higher proviral burden in symptomatic patients, and patients without symptoms, also had higher levels. Possibly, the polymorphism, related to the lower expression of SAMHD1, could promote less inhibition of reverse transcription, leading to the formation of few reverse transcription intermediates (RTIs) and low type I interferon production, resulting in a more productive infection, with a high proviral load  polymorphism is not able to induce the progression and worsening of the infection, but it would act as a factor that could increase the proviral load and contribute to the appearance of symptoms. Other studies, including the follow-up of asymptomatic patients with polymorphism, may better clarify its influence on the development of symptoms associated with HTLV-1.The findings found in the present study suggest that only SAMHD1 rs6029941 (A/G) polymorphism is associated with increased HTLV-1 proviral load and lower levels of IFN-α in symptomatic patients. Thus, the polymorphism could contribute to the development of the symptoms of the disease.In summary, the results suggest that the The datasets used and analyzed during the current study are available from the corresponding author on reasonable requests from a qualified researcher.The studies involving human participants were reviewed and approved by the Research Ethics Committee of the Health Science Institute of the Federal University of Pará (protocol no. 2872434/2018). The patients/participants provided their written informed consent to participate in this study.MQ and EA designed the study. TM, CC, and MS provided technical assistance and executed the experiments. MQ, EA, SL, and AV analyzed and interpreted the date. MQ, RI, and AV wrote the manuscript with input from all authors. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.