A novel Mediterranean diet-inspired supplement ameliorates cognitive, microbial, and metabolic deficits in a mouse model of low-grade inflammation.

The Mediterranean diet (MD) and its bioactive constituents have been advocated for their neuroprotective properties along with their capacity to affect gut microbiota speciation and metabolism. Mediated through the gut brain axis, this modulation of the microbiota may partly contribute to the neuroprotective properties of the MD. To explore this potential interaction, we evaluated the neuroprotective properties of a novel bioactive blend (Neurosyn240) resembling the Mediterranean diet in a rodent model of chronic low-grade inflammation. Behavioral tests of cognition, brain proteomic analysis, 16S rRNA sequencing, and 1H NMR metabolomic analyses were employed to develop an understanding of the gut-brain axis interactions involved. Recognition memory, as assessed by the novel object recognition task (NOR), decreased in response to LPS insult and was restored with Neurosyn240 supplementation. Although the open field task performance did not reach significance, it correlated with NOR performance indicating an element of anxiety related to this cognitive change. Behavioral changes associated with Neurosyn240 were accompanied by a shift in the microbiota composition which included the restoration of the Firmicutes: Bacteroidota ratio and an increase in Muribaculum, Rikenellaceae Alloprevotella, and most notably Akkermansia which significantly correlated with NOR performance. Akkermansia also correlated with the metabolites 5-aminovalerate, threonine, valine, uridine monophosphate, and adenosine monophosphate, which in turn significantly correlated with NOR performance. The proteomic profile within the brain was dramatically influenced by both interventions, with KEGG analysis highlighting oxidative phosphorylation and neurodegenerative disease-related pathways to be modulated. Intriguingly, a subset of these proteomic changes simultaneously correlated with Akkermansia abundance and predominantly related to oxidative phosphorylation, perhaps alluding to a protective gut-brain axis interaction. Collectively, our results suggest that the bioactive blend Neurosyn240 conferred cognitive and microbiota resilience in response to the deleterious effects of low-grade inflammation.

Polymorphism Pro64His within galectin-3 has functional consequences at proteome level in thyroid cells.

Introduction: The single nucleotide polymorphism (SNP) rs4644 at codon 64 of galectin-3 (gal-3, gene name: LGALS3), specifying the variant proline (P64) to histidine (H64), is known to affect the protein's functions and has been associated with the risk of several types of cancer, including differentiated thyroid carcinoma (DTC). Materials and methods: To deepen our understanding of the biological effects of this SNP, we analyzed the proteome of two isogenic cell lines (NC-P64 vs. NA-H64) derived from the immortalized non-malignant thyrocyte cell line Nthy-Ori, generated through the CRISPR-Cas9 technique to differ by rs4644 genotype. We compared the proteome of these cells to detect differentially expressed proteins and studied their proteome in relation to their transcriptome. Results: Firstly, we found, consistently with previous studies, that gal-3-H64 could be detected as a monomer, homodimer, and heterodimer composed of one cleaved and one uncleaved monomer, whereas gal-3-P64 could be found only as a monomer or uncleaved homodimer. Moreover, results indicate that rs4644 influences the expression of several proteins, predominantly upregulated in NA-H64 cells. Overall, the differential protein expression could be attributed to the altered mRNA expression, suggesting that rs4644 shapes the function of gal-3 as a transcriptional co-regulator. However, this SNP also appeared to affect post-transcriptional regulatory mechanisms for proteins whose expression was oppositely regulated compared to mRNA expression. It is conceivable that the rs4644-dependent activities of gal-3 could be ascribed to the different modalities of self-dimerization. Conclusion: Our study provided further evidence that rs4644 could affect the gal-3 functions through several routes, which could be at the base of differential susceptibility to diseases, as reported in case-control association studies.

Dietary fiber supplementation increases Drosophila melanogaster lifespan and gut microbiota diversity.

Dietary fiber has been shown to have multiple health benefits, including a positive effect on longevity and the gut microbiota. In the present study, Drosophila melanogaster has been chosen as an in vivo model organism to study the health effects of dietary fiber supplementation (DFS). DFS extended the mean half-life of male and female flies, but the absolute lifespan only increased in females. To reveal the underlying mechanisms, we examined the effect of DFS on gut microbiota diversity and abundance, local gut immunity, and the brain proteome. A significant difference in the gut microbial community was observed between groups with and without fiber supplementation, which reduced the gut pathogenic bacterial load. We also observed an upregulated expression of dual oxidase and a modulated expression of Attacin and Diptericin genes in the gut of older flies, possibly delaying the gut dysbiosis connected to the age-related gut immune dysfunction. Brain proteome analysis showed that DFS led to the modulation of metabolic processes connected to mitochondrial biogenesis, the RhoV-GTPase cycle, organelle biogenesis and maintenance, membrane trafficking and vesicle-mediated transport, possibly orchestrated through a gut-brain axis interaction. Taken together, our study shows that DFS can prolong the half-life and lifespan of flies, possibly by promoting a healthier gut environment and delaying the physiological dysbiosis that characterizes the ageing process. However, the RhoV-GTPase cycle at the brain level may deserve more attention in future studies.

A Proteomic Approach Identified TFEB as a Key Player in the Protective Action of Novel CB2R Bitopic Ligand FD22a against the Deleterious Effects Induced by ß-Amyloid in Glial Cells.

Neurodegenerative diseases (NDDs) are progressive multifactorial disorders of the nervous system sharing common pathogenic features, including intracellular misfolded protein aggregation, mitochondrial deficit, and inflammation. Taking into consideration the multifaceted nature of NDDs, development of multitarget-directed ligands (MTDLs) has evolved as an attractive therapeutic strategy. Compounds that target the cannabinoid receptor type II (CB2R) are rapidly emerging as novel effective MTDLs against common NDDs, such as Alzheimer's disease (AD). We recently developed the first CB2R bitopic/dualsteric ligand, namely FD22a, which revealed the ability to induce neuroprotection with fewer side effects. To explore the potential of FD22a as a multitarget drug for the treatment of NDDs, we investigated here its ability to prevent the toxic effect of ß-amyloid (Aß25-35 peptide) on human cellular models of neurodegeneration, such as microglia (HMC3) and glioblastoma (U87-MG) cell lines. Our results displayed that FD22a efficiently prevented Aß25-35 cytotoxic and proinflammatory effects in both cell lines and counteracted ß-amyloid-induced depression of autophagy in U87-MG cells. Notably, a quantitative proteomic analysis of U87-MG cells revealed that FD22a was able to potently stimulate the autophagy-lysosomal pathway (ALP) by activating its master transcriptional regulator TFEB, ultimately increasing the potential of this novel CB2R bitopic/dualsteric ligand as a multitarget drug for the treatment of NDDs.

Integrative neuro-cardiovascular dynamics in response to test anxiety: A brain-heart axis study.

Test anxiety (TA), a recognized form of social anxiety, is the most prominent cause of anxiety among students and, if left unmanaged, can escalate to psychiatric disorders. TA profoundly impacts both central and autonomic nervous systems, presenting as a dual manifestation of cognitive and autonomic components. While limited studies have explored the physiological underpinnings of TA, none have directly investigated the intricate interplay between the CNS and ANS in this context. In this study, we introduce a non-invasive, integrated neuro-cardiovascular approach to comprehensively characterize the physiological responses of 27 healthy subjects subjected to test anxiety induced via a simulated exam scenario. Our experimental findings highlight that an isolated analysis of electroencephalographic and heart rate variability data fails to capture the intricate information provided by a brain-heart axis assessment, which incorporates an analysis of the dynamic interaction between the brain and heart. With respect to resting state, the simulated examination induced a decrease in the neural control onto heartbeat dynamics at all frequencies, while the studying condition induced a decrease in the ascending heart-to-brain interplay at EEG oscillations up to 12Hz. This underscores the significance of adopting a multisystem perspective in understanding the complex and especially functional directional mechanisms underlying test anxiety.

Proteomic Profiling Reveals Specific Molecular Hallmarks of the Pig Claustrum.

The present study, employing a comparative proteomic approach, analyzes the protein profile of pig claustrum (CLA), putamen (PU), and insula (IN). Pig brain is an interesting model whose key translational features are its similarities with cortical and subcortical structures of human brain. A greater difference in protein spot expression was observed in CLA vs PU as compared to CLA vs IN. The deregulated proteins identified in CLA resulted to be deeply implicated in neurodegenerative (i.e., sirtuin 2, protein disulfide-isomerase 3, transketolase) and psychiatric (i.e., copine 3 and myelin basic protein) disorders in humans. Metascape analysis of differentially expressed proteins in CLA vs PU comparison suggested activation of the α-synuclein pathway and L1 recycling pathway corroborating the involvement of these anatomical structures in neurodegenerative diseases. The expression of calcium/calmodulin-dependent protein kinase and dihydropyrimidinase like 2, which are linked to these pathways, was validated using western blot analysis. Moreover, the protein data set of CLA vs PU comparison was analyzed by Ingenuity Pathways Analysis to obtain a prediction of most significant canonical pathways, upstream regulators, human diseases, and biological functions. Interestingly, inhibition of presenilin 1 (PSEN1) upstream regulator and activation of endocannabinoid neuronal synapse pathway were observed. In conclusion, this is the first study presenting an extensive proteomic analysis of pig CLA in comparison with adjacent areas, IN and PUT. These results reinforce the common origin of CLA and IN and suggest an interesting involvement of CLA in endocannabinoid circuitry, neurodegenerative, and psychiatric disorders in humans.

Dietary Supplementation with Boswellia serrata, Verbascum thapsus, and Curcuma longa in Show Jumping Horses: Effects on Serum Proteome, Antioxidant Status, and Anti-Inflammatory Gene Expression.

Intense exercise can cause inflammation and oxidative stress due to the production of reactive oxygen species. These pathophysiological processes are interdependent, and each one can induce the other, creating a vicious circle. A placebo-controlled blind study was carried out in show jumping horses (n. 16) to evaluate the effects of a commercial dietary supplement (Dolhorse® N.B.F. Lanes srl, Milan, Italy) containing Verbascum thapsus leaf powder (1.42%), Curcuma longa (14.280 mg/kg), and Boswellia serrata (Roxb ex Colebr) (14.280 mg/kg) extracts. Before and after 10 days of dietary supplementation, blood samples were collected to evaluate the protein levels, antioxidants, and inflammatory responses by proteomic analysis or real-time Reverse Transcriptase-Polymerase Chain Reaction (real-time RT-PCR). A total of 36 protein spots, connected to 29 proteins, were modulated by dietary supplementation, whereas real-time RT-PCR revealed a significant downregulation of proinflammatory cytokines (interleukin 1α (p < 0.05) and interleukin-6 (0.005), toll-like receptor 4 (p < 0.05), and IKBKB (p < 0.05) in supplemented sport horses. Immunoglobulin chains, gelsolin, plasminogen, vitamin D binding protein, apolipoprotein AIV, and filamin B were overexpressed, whereas haptoglobin, α-2-HS-glycoprotein, α2-macroglobulin, afamin, amine oxidase, 60S acidic ribosomal protein, and complement fragments 3, 4, and 7 were reduced. No effect was observed on the antioxidant defense systems. The present results suggest this phytotherapy may reinforce the innate immune responses, thus representing a valid adjuvant to alleviate inflammation, which is a pathophysiological process in sport horses.

Saffron extract (Safr'Inside™) improves anxiety related behaviour in a mouse model of low-grade inflammation through the modulation of the microbiota and gut derived metabolites.

Treatment of anxiety and depression predominantly centres around pharmacological interventions, which have faced criticism for their associated side effects, lack of efficacy and low tolerability. Saffron, which is reportedly well tolerated in humans, has been recognised for its antidepressant and anti-anxiety properties. Indeed, we previously reported upon the efficacy of saffron extract supplementation in healthy adults with subclinical anxiety. However, the molecular aetiology remains unclear. In a rodent model of low-grade chronic inflammation, we explored the impact of a saffron extract (Safr'Inside™) supplemented at a physiological dose, which equated to 22 ± 1.2 mg per day human equivalent dose for a person of 60 kg. Behavioural tests (Open Field task, Y maze, Novel object recognition), caecal 16S rRNA microbial sequencing, caecal 1H NMR metabolomic analysis and 2DE brain proteomic analyses were completed to probe gut-brain axis interactions. Time occupying the centre of the Open Field maze (OF) was increased by 62% in saffron supplemented animals. This improvement in anxiety-related behaviour coincided with gut microbial shifts, notably Akkermansia, Muribaculaceae, Christensenellacae and Alloprevotella which significantly increased in response to saffron supplementation. Akkermansia and Muribaculaceae abundance negatively correlated with the neurotoxic metabolite dimethylamine which was reduced in saffron supplemented animals. Brain proteomic analysis highlighted several significantly altered proteins including ketimine reductase mu-crystallin which also correlated with dimethylamine concentration. Both dimethylamine and ketimine reductase mu-crystallin were associated with OF performance. This may be indicative of a novel interaction across the gut-brain axis which contributes to anxiety-related disorders.

Role of Prosaposin and Extracellular Sulfatase Sulf-1 Detection in Pleural Effusions as Diagnostic Biomarkers of Malignant Mesothelioma.

Malignant pleural mesothelioma is an aggressive malignancy with poor prognosis. Unilateral pleural effusion is frequently the initial clinical sign requiring therapeutic thoracentesis, which also offers a diagnostic opportunity. Detection of soluble biomarkers can support diagnosis, but few show good diagnostic accuracy. Here, we studied the expression levels and discriminative power of two putative biomarkers, prosaposin and extracellular sulfatase SULF-1, identified by proteomic and transcriptomic analysis, respectively. Pleural effusions from a total of 44 patients (23 with mesothelioma, 8 with lung cancer, and 13 with non-malignant disease) were analyzed for prosaposin and SULF-1 by enzyme-linked immunosorbent assay. Pleural effusions from mesothelioma patients had significantly higher levels of prosaposin and SULF-1 than those from non-malignant disease patients. Receiver-operating characteristic (ROC) analysis showed that both biomarkers have good discriminating power as pointed out by an AUC value of 0.853 (p = 0.0005) and 0.898 (p < 0.0001) for prosaposin and SULF-1, respectively. Combining data ensued a model predicting improvement of the diagnostic performance (AUC = 0.916, p < 0.0001). In contrast, prosaposin couldn't discriminate mesothelioma patients from lung cancer patients while ROC analysis of SULF-1 data produced an AUC value of 0.821 (p = 0.0077) but with low sensitivity. In conclusion, prosaposin and SULF-1 levels determined in pleural effusion may be promising biomarkers for differential diagnosis between mesothelioma and non-malignant pleural disease. Instead, more patients need to be enrolled before granting the possible usefulness of these soluble proteins in differentiating mesothelioma pleural effusions from those linked to lung cancer.

The Protective Action of Metformin against Pro-Inflammatory Cytokine-Induced Human Islet Cell Damage and the Mechanisms Involved.

Metformin, a drug widely used in type 2 diabetes (T2D), has been shown to protect human ß-cells exposed to gluco- and/or lipotoxic conditions and those in islets from T2D donors. We assessed whether metformin could relieve the human ß-cell stress induced by pro-inflammatory cytokines (which mediate ß-cells damage in type 1 diabetes, T1D) and investigated the underlying mechanisms using shotgun proteomics. Human islets were exposed to 50 U/mL interleukin-1ß plus 1000 U/mL interferon-γ for 48 h, with or without 2.4 µg/mL metformin. Glucose-stimulated insulin secretion (GSIS) and caspase 3/7 activity were studied, and a shotgun label free proteomics analysis was performed. Metformin prevented the reduction of GSIS and the activation of caspase 3/7 induced by cytokines. Proteomics analysis identified more than 3000 proteins in human islets. Cytokines alone altered the expression of 244 proteins (145 up- and 99 down-regulated), while, in the presence of metformin, cytokine-exposure modified the expression of 231 proteins (128 up- and 103 downregulated). Among the proteins inversely regulated in the two conditions, we found proteins involved in vesicle motility, defense against oxidative stress (including peroxiredoxins), metabolism, protein synthesis, glycolysis and its regulation, and cytoskeletal proteins. Metformin inhibited pathways linked to inflammation, immune reactions, mammalian target of rapamycin (mTOR) signaling, and cell senescence. Some of the changes were confirmed by Western blot. Therefore, metformin prevented part of the deleterious actions of pro-inflammatory cytokines in human ß-cells, which was accompanied by islet proteome modifications. This suggests that metformin, besides use in T2D, might be considered for ß-cell protection in other types of diabetes, possibly including early T1D.

Pterostilbene Promotes Mean Lifespan in Both Male and Female Drosophila Melanogaster Modulating Different Proteins in the Two Sexes.

Aging is a multifactorial phenomenon characterized by degenerative processes closely connected to oxidative damage and chronic inflammation. Recently, many studies have shown that natural bioactive compounds are useful in delaying the aging process. In this work, we studied the effects of an in vivo supplementation of the stilbenoid pterostilbene on lifespan extension in Drosophila melanogaster. We found that the average lifespan of flies of both sexes was increased by pterostilbene supplementation with a higher effect in females. The expression of longevity related genes (Sir2, Foxo, and Notch) was increased in both sexes but with different patterns. Pterostilbene counteracted oxidative stress induced by ethanol and paraquat and up-regulated the antioxidant enzymes Ho e Trxr-1 in male but not in female flies. On the other hand, pterostilbene decreased the inflammatory mediators dome and egr only in female flies. Proteomic analysis revealed that pterostilbene modulates 113 proteins in male flies and only 9 in females. Only one of these proteins was modulated by pterostilbene in both sexes: vacuolar H[+] ATPase 68 kDa subunit 2 (Vha68-2) that was strongly down-regulated. These findings suggest a potential role of pterostilbene in increasing lifespan both in male and female flies by mechanisms that seem to be different in the two sexes, highlighting the need to conduct nutraceutical supplementation studies on males and females separately in order to give more reliable results.

Parathyroid Carcinoma and Adenoma Co-existing in One Patient: Case Report and Comparative Proteomic Analysis.

BACKGROUND/AIM: The lack of specific parathyroid carcinoma (PC) biomarkers in clinical practice points out the importance of analyzing the proteomic signature of this cancer. We performed a comparative proteomic analysis of PC and parathyroid adenoma (PA) co-existing in the same patient. PATIENTS AND METHODS: PC and PA were taken from a 63-year-old patient. Using two-dimensional differential gel electrophoresis (2D-DIGE) coupled to mass spectrometry we examined the differences between PC and PA proteins. For validation, additional PC and PA samples were obtained from 10 patients. Western blot analysis was used to validate the difference of expression observed with 2D-DIGE analysis. Bioinfomatic analysis was performed using QIAGEN's Ingenuity Pathways Analysis (IPA) to determine the predominant canonical pathways and interaction networks involved. RESULTS: Thirty-three differentially expressed proteins were identified in PC compared to PA. Among these, ubiquitin C-terminal hydrolase-L1 (UCH-L1) was highly overexpressed in PC. The result was confirmed by Western Blot analysis in additional PC samples. CONCLUSION: Our comparative proteomic analysis of co-existing neoplasms allowed detecting specific and peculiar differences between PC and PA overcoming population biological variability.

Age-Related Changes in the Primary Motor Cortex of Newborn to Adult Domestic Pig Sus scrofa domesticus.

The pig has been increasingly used as a suitable animal model in translational neuroscience. However, several features of the fast-growing, immediately motor-competent cerebral cortex of this species have been adequately described. This study analyzes the cytoarchitecture of the primary motor cortex (M1) of newborn, young and adult pigs (Sus scrofa domesticus). Moreover, we investigated the distribution of the neural cells expressing the calcium-binding proteins (CaBPs) (calretinin, CR; parvalbumin, PV) throughout M1. The primary motor cortex of newborn piglets was characterized by a dense neuronal arrangement that made the discrimination of the cell layers difficult, except for layer one. The absence of a clearly recognizable layer four, typical of the agranular cortex, was noted in young and adult pigs. The morphometric and immunohistochemical analyses revealed age-associated changes characterized by (1) thickness increase and neuronal density (number of cells/mm2 of M1) reduction during the first year of life; (2) morphological changes of CR-immunoreactive neurons in the first months of life; (3) higher density of CR- and PV-immunopositive neurons in newborns when compared to young and adult pigs. Since most of the present findings match with those of the human M1, this study strengthens the growing evidence that the brain of the pig can be used as a potentially valuable translational animal model during growth and development.

Preclinical Development of FA5, a Novel AMP-Activated Protein Kinase (AMPK) Activator as an Innovative Drug for the Management of Bowel Inflammation.

Acadesine (ACA), a pharmacological activator of AMP-activated protein kinase (AMPK), showed a promising beneficial effect in a mouse model of colitis, indicating this drug as an alternative tool to manage IBDs. However, ACA displays some pharmacodynamic limitations precluding its therapeutical applications. Our study was aimed at evaluating the in vitro and in vivo effects of FA-5 (a novel direct AMPK activator synthesized in our laboratories) in an experimental model of colitis in rats. A set of experiments evaluated the ability of FA5 to activate AMPK and to compare the efficacy of FA5 with ACA in an experimental model of colitis. The effects of FA-5, ACA, or dexamethasone were tested in rats with 2,4-dinitrobenzenesulfonic acid (DNBS)-induced colitis to assess systemic and tissue inflammatory parameters. In in vitro experiments, FA5 induced phosphorylation, and thus the activation, of AMPK, contextually to the activation of SIRT-1. In vivo, FA5 counteracted the increase in spleen weight, improved the colon length, ameliorated macroscopic damage score, and reduced TNF and MDA tissue levels in DNBS-treated rats. Of note, FA-5 displayed an increased anti-inflammatory efficacy as compared with ACA. The novel AMPK activator FA-5 displays an improved anti-inflammatory efficacy representing a promising pharmacological tool against bowel inflammation.

Antioxidant and Neuroprotective Activity of Extra Virgin Olive Oil Extracts Obtained from Quercetano Cultivar Trees Grown in Different Areas of the Tuscany Region (Italy).

Neurodegenerative diseases are driven by several mechanisms such as inflammation, abnormal protein aggregation, excitotoxicity, mitochondrial dysfunction and oxidative stress. So far, no therapeutic strategies are available for neurodegenerative diseases and in recent years the research is focusing on bioactive molecules present in food. In particular, extra-virgin olive oil (EVOO) phenols have been associated to neuroprotection. In this study, we investigated the potential antioxidant and neuroprotective activity of two different EVOO extracts obtained from Quercetano cultivar trees grown in two different areas (plain and hill) of the Tuscany region (Italy). The different geographical origin of the orchards influenced phenol composition. Plain extract presented a higher content of phenyl ethyl alcohols, cinnammic acids, oleacein, oleocanthal and flavones; meanwhile, hill extract was richer in lignans. Hill extract was more effective in protecting differentiated SH-SY5Y cells from peroxide stress thanks to a marked upregulation of the antioxidant enzymes heme oxygenase 1, NADPH quinone oxidoreductase 1, thioredoxin Reductase 1 and glutathione reductase. Proteomic analysis revealed that hill extract plays a role in the regulation of proteins involved in neuronal plasticity and activation of neurotrophic factors such as BDNF. In conclusion, these data demonstrate that EVOOs can have important neuroprotective activities, but these effects are strictly related to their specific phenol composition.

Salivary Proteome Changes in Response to Acute Psychological Stress Due to an Oral Exam Simulation in University Students: Effect of an Olfactory Stimulus.

The autonomic nervous system (ANS) plays a crucial role both in acute and chronic psychological stress eliciting changes in many local and systemic physiological and biochemical processes. Salivary secretion is also regulated by ANS. In this study, we explored salivary proteome changes produced in thirty-eight University students by a test stress, which simulated an oral exam. Students underwent a relaxation phase followed by the stress test during which an electrocardiogram was recorded. To evaluate the effect of an olfactory stimulus, half of the students were exposed to a pleasant odor diffused in the room throughout the whole session. Saliva samples were collected after the relaxation phase (T0) and the stress test (T1). State anxiety was also evaluated at T0 and T1. Salivary proteins were separated by two-dimensional electrophoresis, and patterns at different times were compared. Spots differentially expressed were trypsin digested and identified by mass spectrometry. Western blot analysis was used to validate proteomic results. Anxiety scores and heart rate changes indicated that the fake exam induced anxiety. Significant changes of α-amylase, polymeric immunoglobulin receptor (PIGR), and immunoglobulin α chain (IGHA) secretion were observed after the stress test was performed in the two conditions. Moreover, the presence of pleasant odor reduced the acute social stress affecting salivary proteome changes. Therefore, saliva proteomic analysis was a useful approach to evaluate the rapid responses associated to an acute stress test also highlighting known biomarkers.

Pro64His (rs4644) Polymorphism Within Galectin-3 Is a Risk Factor of Differentiated Thyroid Carcinoma and Affects the Transcriptome of Thyrocytes Engineered via CRISPR/Cas9 System.

Background: Galectin-3 (LGALS3) is an important glycoprotein involved in the malignant transformation of thyrocytes acting in the extracellular matrix, cytoplasm, and nucleus where it regulates TTF-1 and TCF4 transcription factors. Within LGALS3 gene, a common single-nucleotide polymorphism (SNP) (c.191C>A, p.Pro64His; rs4644) encoding for the variant Proline to Histidine at codon 64 has been extensively studied. However, data on rs4644 in the context of thyroid cancer are lacking. Thus, the aim of the present work was to evaluate the role of the rs4644 SNP as risk factor for differentiated thyroid cancer (DTC) and to determine the effect on the transcriptome in thyrocytes. Methods: A case/control association study in 1223 controls and 1142 unrelated consecutive DTC patients was carried out to evaluate the association between rs4644-P64H and the risk of DTC. We used the nonmalignant cell line Nthy-Ori (rs4644-C/A) and the CRISPR/Cas9 technique to generate isogenic cells carrying either the rs4644-A/A or rs4644-C/C homozygosis. Then, the transcriptome of the derivative and unmodified parental cells was analyzed by RNA-seq. Genes differentially expressed were validated by quantitative reverse transcription PCR and further tested in the parental Nthy-Ori cells after LGALS3 gene silencing, to investigate whether the expression of target genes was dependent on galectin-3 levels. Results: rs4644 AA genotype was associated with a reduced risk of DTC (compared with CC, ORadj = 0.66; 95% confidence interval = 0.46-0.93; Pass = 0.02). We found that rs4644 affects galectin-3 as a transcriptional coregulator. Among 34 genes affected by rs4644, HES1, HSPA6, SPC24, and NHS were of particular interest since their expression was rs4644-dependent (CC>AA for the first and AA>CC for the others), also in 574 thyroid tissues of Genotype-Tissue Expression (GTEx) biobank. Moreover, the expression of these genes was regulated by LGALS3-silencing. Using the proximity ligation assay in Nthy-Ori cells, we found that the TTF-1 interaction was genotype dependent. Conclusions: Our data show that in thyroid, rs4644 is a trans-expression quantitative trait locus that can modify the transcriptional expression of downstream genes, through the modulation of TTF-1.