The Insulin-like growth factor (IGF) axis is one of the best-established drivers of thyroid transformation, as thyroid cancer cells overexpress both IGF ligands and their receptors. Thyroid neoplasms encompass distinct clinical and biological entities as differentiated thyroid carcinomas (DTC)—comprising papillary (PTC) and follicular (FTC) tumors—respond to radioiodine therapy, while undifferentiated tumors—including poorly-differentiated (PDTC) or anaplastic thyroid carcinomas (ATCs)—are refractory to radioactive iodine and exhibit limited responses to chemotherapy. Thus, safe and effective treatments for the latter aggressive thyroid tumors are urgently needed. Despite a strong preclinical rationale for targeting the IGF axis in thyroid cancer, the results of the available clinical studies have been disappointing, possibly because of the crosstalk between IGF signaling and other pathways that may result in resistance to targeted agents aimed against individual components of these complex signaling networks. Based on these observations, the combinations between IGF-signaling inhibitors and other anti-tumor drugs, such as DNA damaging agents or kinase inhibitors, may represent a promising therapeutic strategy for undifferentiated thyroid carcinomas. In this review, we discuss the role of the IGF axis in thyroid tumorigenesis and also provide an update on the current knowledge of IGF-targeted combination therapies for thyroid cancer. Insulin-like growth factor (IGF) signaling promotes cell proliferation and differentiation in normal human tissues ,2.The IGF system comprises the IGF ligands 1 (IGF-1) and 2 (IGF-2) which modulate multiple biological processes by interacting with specific cell-surface receptors represented by IGF receptors I and II (IGF-IR and IGF-IIR) ,4,5,6. IIGF-IR and IGF-IIR (IGFRs) show different structures and function. In detail, IGF-IR is a member of the receptor tyrosine kinases (RTK) and binds IGF-1 with a higher affinity than IGF-2. IGF-IR forms a tetrameric complex consisting of two alpha and two beta subunits linked by disulfide bonds through fibronectin type III (FnIII) domains. The alpha subunit localizes in the extracellular space and consists of two large homologous regions (L1 and L2) separated by a cysteine rich domain responsible for ligand binding. The beta subunit displays a trans-membrane segment and an intracellular portion containing the tyrosine kinase domain (TKD) that actively phosphorylates direct downstream targets on selected tyrosine residues . Unlike An additional feature of the IGF axis is its crosstalk with insulin and insulin receptors isoforms A (IR-A) and B (IR-B). Indeed, the IGF system contributes to thyroid cancerogenesis via an IGF-2/IR-A autocrine loop as IGF-2 activates the IR-A isoform over-expressed in neoplastic thyrocytes, thus promoting their proliferation and suppressing apoptosis ,10. HoweTo date, several studies have established that aberrant IGF signaling plays a critical role in cancer pathogenesis and progression. Increased expression of IGF ligands and receptors has been observed in tumors of the breast, lung, pancreas, colon, prostate, ovary, and thyroid and is usually associated with a poor prognosis ,6,14,15.Thyroid cancer is a frequently encountered endocrine malignancy sometimes aided by exposure to multiple environmental carcinogens favoring the generation of different molecular alterations ,18,19,20As the IGF system has been actively investigated in thyroid cancer ,10,14,23Deregulation of the IGF axis is emerging as a key factor promoting proliferative responses in both normal and neoplastic thyrocytes ,10,24,25Since the IGF cascade relies on IGFBP-mediated interactions between the IGF ligands and IGF-IR, several studies have investigated if IGFBPs are involved in thyroid tumorigenesis ,30. IndeSignaling downstream of the IGF axis involves intracellular mediators contributing to cell proliferation Rat Sarcoma (RAS)/Proto-Oncogene Serine/Threonine-Protein Kinase (RAF)/Mitogen-Activated Protein Kinase inhibitors (MEK)/Extracellular Signal-Regulated Kinase (ERK), apoptosis inhibition, protein synthesis, cell cycle progression Phosphatidylinositol kinase (PI3K)/V-Akt Murine Thymoma Viral Oncogene Homolog (AKT)/Mammalian Target of Rapamycin (mTOR) and cell motility Focal Adhesion Kinase (FAK) ,34,35. AThe involvement of the IGF axis in thyroid carcinoma is also related to crosstalk interactions between the IGF-IR and thyroid-stimulating hormone (TSH). In fact, the pro-tumorigenic effect of TSH is reduced in the absence of growth factors, but is increased by IGF-1 stimulation ,39,40. MMET), the discoidin domain receptor tyrosine kinase 1 (DDR1) and the anaplastic lymphoma kinase 1 (ALK) [An additional layer of complexity to this already composite signaling network is represented by the well documented crosstalk between the IGF axis and several RTKs including the insulin receptor (IR), epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), hepatocyte growth factor receptor (HGFR-proto-oncogene  1 (ALK) ,45,46,47 1 (ALK) ,49,50,51IGF-IR direct inhibitors) or indirectly (IGF-IR/RTKs downstream inhibitors) interfere with the IGF axis in thyroid carcinoma  and tyrosine kinase inhibitors (TKIs) binding to the IGF-IR catalytic domain (IGF-IRTKIs) [mAbs and IGF-IRTKIs with different anticancer drugs.Agents exerting IGF-IR inhibition include monoclonal antibodies (mAbs) targeting IGF-IR (IGF-IR-IRTKIs) . Disappo-IRTKIs) . Thus, fA comprehensive description of their possible use in the preclinical or clinical settings both, in monotherapy or in combination with additional pharmacological compounds is included below.mAbs block ligand–receptor interactions, causing receptor internalization and degradation and thereby quenching IGF-IR-mediated intracellular signaling. Several IGF-IRmAbs have been generated and tested in different tumor types [IGF-IRor types  but onlyA phase I study evaluated the efficacy of the combination of AVE1642 with docetaxel in a cohort of patients affected by different tumor types including one patient with thyroid carcinoma. More than 50% of subjects enrolled in this group achieved stable disease .Preclinical studies evaluated the efficacy of cixitumumab both in vitro and in vivo using an orthotopic mouse model of ATC . In thisA phase Ib basket trial investigated dual treatment with ganitumab and sorafenib or panitumumab in two patients with thyroid carcinoma . In thismAbs as anticancer agents, an important implication concerning their adverse events profile (AEs) must be considered. These pitfalls were reported in different clinical studies and include severe AEs such as early fatal toxicities [mAbs as used as anticancer agents, it is essential apply the correct combination with other anticancer drugs in order to reduce the AEs.Despite the attractive role of IGF-IRxicities , hyper- xicities . Hence, TKIs are small molecules that bind and functionally inhibit the IGF-IR catalytic domain. Currently, only results with linsitinib, NVP-ADW742, and NVP-AEW541 have been reported.IGF-IRThe biological cooperation between TSH and IGF-IR was investigated in primary human thyrocytes where linsitinib inhibited TSH-mediated NIS stimulation that requires ERK and/or AKT signaling. These data highlight the importance of the TSH/IGF-IR crosstalk in thyroid cancer .Constantine and colleagues evaluated the efficacy of NVP-ADW742 against multiple tumor cell types including thyroid carcinoma cell lines. They observed that NVP-ADW742 reduces IGF-IR phosphorylation after IGF-I stimulation and inhibits cell proliferation .WRO thyroid cancer cells genetically modified to express the IGF-IR were implanted in thyroid glands of athymic mice and exposed to NVP-AEW541 or irinotecan, used as a reference treatment. NVP-AEW541 inhibited tumor growth more potently than irinotecan. Furthermore, NVP-AEW541 also reduced tumor angiogenesis, suggesting that IGF-IR modulates thyroid tumor microenvironment .A further therapeutic strategy examined in thyroid carcinomas relies on agents that interfere with the complex downstream signaling generated by the crosstalk between IGF-IR and other RTKs ,51,87,88Aberrant activation of PI3K/AKT/mTOR signaling heavily contributes to thyroid tumorigenesis ,90,91. HA phase I and a phase II clinical trial enrolling patients with refractory FTC and PDTC failed to detect reductions in tumor growth after buparlisib treatment ,65. ThesA phase I study tested the drug’s efficacy in different tumor types including one patient with thyroid carcinoma. No results have yet been reported .This mTOR inhibitor generated contrasting results when employed on thyroid tumors as efficacy was mostly dependent on tumor type and stage. Indeed, while low response rates were reported in patients with locally advanced or metastatic thyroid cancer , everoliA retrospective study evaluated the efficacy of sirolimus and cyclophosphamide in patients with advanced differentiated thyroid carcinoma. The association was well tolerated, but no differences in progression-free survival were detected between patients in the experimental arm and those allocated to the control group (receiving standard of care for disease progression) .The ability of temsirolimus to interfere with thyroid tumor growth was described in association with anti-angiogenic agent (trebaninib) or multikinase inhibitor (sorafenib). Specifically, a phase I trial enrolling patients with different tumor types including one with thyroid carcinoma found that the association of temsirolimus and trebaninib induced a partial response . A more Aberrant activation of intracellular signaling involving the MEK pathway contributes to thyroid tumorigenesis since MEK inhibition favors differentiation of radioiodine-refractory DTCs by restoring NIS expression ,95,96. HTwo phase I studies investigated the efficacy of binimetinib and the association of pimasertib with temsirolimus in patients with advanced solid tumors including thyroid cancer. However, no data have yet been reported with the exception of a case report describing a patient receiving binimetinib that achieved a partial response (tumor shrinkage >30%) ,70.B-RAFV600E mutation [In a clinical trial (NCT009700359) enrolling 24 patients with thyroid cancer refractory to radioiodine, selumetinib generated clinically meaningful increases in iodine uptake . This immutation .This compound is an allosteric MEK inhibitor with documented anti-tumor activity against thyroid cancer. A phase I dose-escalating basket trial evaluated trametinib efficacy in five patients with thyroid carcinomas. All patients achieved stable disease or partial responses . A phaseAlthough both in vitro and in vivo models have demonstrated an active role for FAK in thyroid tumorigenesis ,103,104,In the last few years, the incidence of thyroid carcinoma is increasing across multiple countries . AlthougIncreased understanding of cancer biology has led to the development of anticancer drugs targeting specific oncogenic substrates such as AKT, BCR-ABL1, EGFR, MET, and the IGF-IR. Since the IGF system is often deregulated in thyroid cancer, it has been considered an attractive pharmacological target for this disease . HoweverIn this complex scenario, combined targeted regimens may overcome these resistance phenomena and improve patient response rates ,109,110.Among the most promising pharmacological agents targeting downstream signaling mediators shared by both IGF-IR and RTKs, the combination of anti-mTOR compounds with different kinase inhibitors has generated encouraging reductions in tumor growth . MEK inhIn summary, while multiple pharmacological approaches aimed against the IGF axis have been actively investigated for the treatment of PDTCs and ATCs, further biological studies and additional compounds and/or pharmacological strategies will be required to significantly improve the outcome of these aggressive forms of thyroid cancer.


MicroRNAs (miRNAs or miRs) have emerged as key regulators of gene expression in essential cellular processes, such as cell growth, differentiation and development. Recent findings have established that the levels of miRNAs are modulated by cell signaling mechanisms, including the bone morphogenetic protein (BMP) signaling pathway. The BMP signaling pathway controls diverse cellular activities by modulating the levels of miRNAs, indicating the complexity of gene regulation by the BMP signaling pathway. The tight regulation of the levels of miRNAs is critical for maintaining normal physiological conditions, and dysregulated miRNA levels contribute to the development of diseases. In the present review, we discuss different insights (provided over the past decade) into the regulation of miRNAs governed by the BMP signaling pathway and the implications of this regulation on the understanding of the cellular differentiation of vascular smooth muscle cells (VSMCs), osteoblasts and neuronal cells. Bone morphogenetic proteins (BMPs) are members of the transforming growth factor (TGF)-β superfamily, acting as potent regulators during embryogenesis and controlling such events as vascular development, bone formation and neuronal differentiation . In respMicroRNAs (miRNAs or miRs) are small non-coding RNA molecules evolutionarily conserved from plants to humans . miRNAs In the present review, we discuss and summarize the miRNAs that are mediated by the BMP signaling pathway in essential biological processes involving vascular smooth muscle cell (VSMC) differentiation, osteogenesis and neuronal development .The inactivation of the BMP signaling pathway has been shown to result in the development of vascular disorders . For exaUpon TGF-β and BMP signaling, Smads interact with p68 in the Drosha microprocessor complex and promote the cleavage of pri-miRNA-21 into pre-miRNA-21, leading to an increase in the levels of mature miRNA-21 in VSMCs . The incIn addition to miRNA-21, TGF-β and BMP signals modulate the expression of a subset of miRNAs through Smad-mediated post-transcriptional regulation . NotablyThe regulation of miRNA-96 expression by BMP signaling is critical for the modulation of the VSMC phenotype . miRNA-9BMP signaling also downregulates transcription of the miRNA-302~367 gene cluster in various types of cells, including VSMCs . This trmiRNA-143 or miRNA-145 knockout mice exhibit an abnormal vascular tone and reduced SMC-specific gene expression in VSMCs, suggesting that miRNA-143 and miRNA-145, which are encoded as a gene cluster, play a critical role in the regulation of the VSMC phenotype . BMP sigin situ hybridization with vessels, suggesting that BMP signaling plays a role in VSMC calcification by regulating miRNAs. Indeed, a microarray analysis demonstrated that BMP2 decreases miRNA-30b and miRNA-30c expression, leading to the promotion of VSMC calcification  into the osteogenic lineage .BMP2, 4 and 7 act as potential differentiators through the Smad-mediated activation of osteoblast essential genes, such as Runx2 . RecentlmiRNA microarray analysis has revealed that miRNA-133 and miRNA-135 are downregulated during the BMP2-induced osteogenesis of C2C12 mesenchymal cells . Both mimiRNA expression in BMP-2-treated mouse pre-osteoblast MC3T3-E1 cells was previously investigated and the downregulation of miRNA-141 and miRNA-200a was observed . miRNA-1OPN), parathyroid hormone-related protein (PTHrP), Runx2 and tenascin-C and type I procollagen  is a potent transcription factor that promotes osteoblast differentiation and bone regeneration. Satb2 functions as a protein scaffold to increase the activity of two essential osteogenic transcription factors, Runx2 and activating transcription factor 4 (ATF4) . The difOsx and other osteogenic genes -mediated proliferation of cerebellar granular neuronal precursors (CGNPs) . To addrThe expression of miRNA-134 has been shown to be increased during embryonic neuronal differentiation and modulates dendritic maturation in response to exogenous BMP4 by targeting the BMP antagonist, Chordin-like 1 (Chrdl-1) . The redThe BMP signaling pathway is involved in many cellular processes, including cell growth and differentiation. BMP signals either upregulate or downregulate a subset of miRNAs, and these coordinately regulated miRNAs cooperate for BMP signaling-mediated cellular functions. Therefore, it is important to understand how cells integrate the complicated regulation of miRNAs whose expression levels are fine-tuned by BMP signaling pathways and transmit a precise signal to control normal development and maintain homeostasis.In the present review, we summarized the regulation of miRNA expression during BMP signaling pathway-mediated cellular differentiation, in particular VSMC differentiation, osteogenesis and neurogenesis. In most reported cases, miRNA levels are regulated by BMP signals, but BMP signaling is able to be regulated by miRNAs through targeting of BMP signal transducers or inhibitory molecules. For example, miRNA-30 and miRNA-133/135 target Smad1 and Smad5, respectively . This inAlthough the differential expression of a subset of miRNAs during BMP signaling-mediated differentiation has been elucidated in various contexts, many factors regulating the cellular miRNA levels are still unknown. Some miRNAs, such as miRNA-302, are transcriptionally regulated by direct binding of Smads to the promoter of the miRNA gene . Alterna


Unexpectedly, some CPPs, (D-Arg)8 and Decanoyl-(D-Arg)8, alone displayed potent antiviral effect, altering late stages of DHBV and HBV morphogenesis. Such antiviral effects of CPPs may affect the sequence-specificity of CPP-PNA conjugates. By contrast, PNA conjugated to (D-Lys)4 inhibited hepadnaviral replication without compromising sequence specificity. Interestingly, Lactose-modified CPP mediated the delivery of anti-HBV PNA to human hepatoma cells HepaRG, thus improving its antiviral activity. In light of these promising data, we believe that future studies will open new perspectives for translation of CPPs and CPP-PNA based technology to therapy of chronic hepatitis B.Alternative therapeutic approaches against chronic hepatitis B virus (HBV) infection need to be urgently developed because current therapies are only virostatic. In this context, cell penetration peptides (CPPs) and their Peptide Nucleic Acids (PNAs) cargoes appear as a promising novel class of biologically active compounds. In this review we summarize different in vitro and in vivo studies, exploring the potential of CPPs as vehicles for intracellular delivery of PNAs targeting hepadnaviral replication. Thus, studies conducted in the duck HBV (DHBV) infection model showed that conjugation of (D-Arg) Hepatitis B virus (HBV) infection is still a major public health issue, with over 240 million virus carriers worldwide. Indeed, chronic HBV infection significantly increases the risk for the development of end stage liver diseases such as cirrhosis and hepatocellular carcinoma (HCC) . CurrentMoreover, the long-term use of these therapies increases the risk of drug resistance and of serious side effects. Therefore, alternative therapeutic approaches need to be urgently developed to eradicate chronic hepatitis B infection.N-(2-aminoethyl)-glycine units to which the nucleobases are attached via methylene carbonyl linkers . Ou. Ou8 waslication .8, different peptide doses were administrated daily to both LMH-D2 and PDH cells. Thus, our results showed that treatment of these cells for seven days by different concentrations of decanoyl-(DArg)8 peptide led to a strong and dose-dependent inhibition of viral secretion reaching 90% at 2 μM (8 is caused only by the cationic (DArg)8 moiety or also by the alkyl chain (decanoic acid). Interestingly, the administration of (DArg)8 CPP led to a dose-dependent decrease of viral secretion reaching 60%, while decanoic acid alone was unable to significantly inhibit DHBV secretion . T. T8, dif8 CPP is considerably lower as compared to that displayed by decanoyl-(DArg)8 peptide in the same experimental conditions, indicating that the amphipathic molecules used to modify CPP sequences are able to improve their cellular uptake and consequently their biological activities. Interestingly, study of intracellular uptake of decanoyl-(DArg)8-Fluorescein isothiocyanate (FITC) and (DArg8)-FITC peptides using fluorescence microscopy, showed that at the same dose, decanoyl-(DArg)8-FITC peptide was internalized more powerfully into the PDH cultures than was (D-Arg8)-FITC. These data are in agreement with the greater antiviral activity of decanoyl-(DArg)8 as compared to the (DArg8) peptide ..8 on HBV8 has a direct impact on DHBV infectivity and the susceptibility of PDH to viral infection, since the cationic antimicrobial peptides are known to induce a strong membrane disruption resulting in an antiviral effect. Our results revealed that decanoyl-(DArg)8 peptide neither directly inactivated DHBV nor interacted with its cellular receptor. Indeed, the pre-incubation of decanoyl-(DArg)8 peptide with a DHBV-positive inoculum or with PDH cells did not diminish virus infectivity and did not affect PDH susceptibility to viral infection [8 peptide is not a ligand of hepadnaviral receptor [Next, we asked whether decanoyl-(DArg)nfection . These rreceptor ,48,49.8 on viral replication to establish whether the strong decrease in DHBV secretion was correlated with an eventual inhibition of DHBV replication. Our data showed that treatment of cells by decanoyl-(DArg)8 led to the accumulation of different viral DNA replicative forms, while this treatment did not affect viral DNA transcription and viral RNA translation. Indeed, decanoyl-(DArg)8 peptide treatment did not modify the intracellular cccDNA synthesis and the expression of the viral envelope and core proteins [Furthermore, we analyzed, in the same PDH and LMH-D2 cell systems, the effect of treatment by decanoyl-(DArg)proteins .8 of PDH and LMH-D2 cells resulted in the formation of large clusters of DHBV envelope proteins, without affecting the intracellular localization of viral core proteins. Moreover, the analysis of DHBV release, using iodixanol-based gradient centrifugation, indicated that decanoyl-(DArg)8 peptide treatment strongly decreased the secretion of both complete virions and subviral particles, while the naked DHBV capsids were still released [Additional studies using confocal laser microscopy scanning revealed that treatment by decanoyl-(DArg)released .8 hampers the envelopment of hepadnaviral capsids. Interestingly, in another report, the synthetic recombinant CPP bearing nucleocapsid binding subunits (CPP-NBS) was shown to efficiently enter into the HepG2.2.15 cells and block viral nucleocapsid assembly, thereby inhibiting the HBV release that was associated with an increase in intracellular hepatitis B core antigen (HBcAg) and HBV DNA replicative intermediates by 2–3 fold [Thus, these findings demonstrate that Decanoyl-(DArg)2–3 fold .8 peptide may indirectly induce severe structural changes in the morphogenesis of the DHBV envelope proteins that may obstruct their association with viral capsids and thus hamper virions secretion [Due to the capacity of CPPs to directly interact with the biologic lipid bilayer of the cells , our datecretion .8, a cationic lipopeptide, is able to strongly interfere with the late stages of hepadnaviral morphogenesis and replication via a drastic blockage of complete virions and subviral particles release [Collectively, our findings support the notion that decanoyl-(DArg) release . This is release . These rAntisense strategies appear to have promise for the control of chronic viral infections. In this view, we initially evaluated the antiviral potential of different antisense molecules such as phosphodiester oligodeoxynucleotides (ODNs), targeting the DHBV large envelope gene. Using the DHBV infection model, we demonstrated that such antisense ODNs complexed to polyethylenimine and administrated to neonatal ducklings via the i.v route were able to drastically inhibit hepadnaviral replication .The pregenomic RNA and hepadnaviral encapsidation signal ε play an important role in initiation of hepadnaviral reverse transcription (RT), and they appear as interesting targets for antiviral therapy ,54. To e8 to increase its cellular uptake. Indeed, the main difficulty in the use of PNAs as antivirals is their poor transport across the cell membrane and their limited intracellular biodistribution. To optimize the in vivo delivery of PNA-CPP conjugates and their bioavailability, we administrated PNA-CPP coupled to FITC or PNA-FITC in vivo to DHBV-free neonatal ducklings. The hepatocyte-associated fluorescence was analyzed by fluorescence microscopy 48 hours later. The results showed little or no hepatocyte-associated fluorescence after i.v injection of FITC-PNA not conjugated to (D-Arg)8, whereas the injection of FITC-CPP-PNA resulted in a high level of hepatocyte-associated fluorescence. This finding indicates that (D-Arg)8 CPP is able to efficiently deliver anti-ε PNA to the liver following intravenous injection  route as only following i.v administration was the FITC-PNA detected in the hepatocytes [Next, to investigate the in vivo antiviral effect of PNAs, we coupled the anti-ε PNA to a cationic CPP (D-Arg)jection  for six days. At the end of this treatment, the analysis of serum and liver DNA revealed a marked decrease in viremia and liver DHBV DNA, i.e. a decrease of about 50% in treated animals compared with the untreated controls (We next asked whether such anti-ε PNA conjugated to (D-Arg)on model . Thus, Dcontrols .8 CPP and used as control also inhibited viral replication. Therefore, an antiviral activity of (D-Arg)8 was suspected. Indeed, the i.v administration of (DArg)8 alone to DHBV-infected ducklings resulted in a decrease in viremia, confirming the ability of this CPP to inhibit hepadnaviral replication (as detailed above). Altogether, these findings indicate that anti-DHBV activity exhibited by (DArg)8 CPP alone may explain the limited sequence specificity of this CPP-PNA conjugate. No loss of weight was observed in all treated animals compared with the untreated control animals during the six days of treatment (data not shown), showing the absence of in vivo toxicity of (DArg)8-PNA conjugates and of (DArg)8 alone.Surprisingly, 2 nt mismatched PNA coupled to the same (DArg)4 in vitro, in PDH. Moreover, the antiviral activity of this (D-Lys)4 CPP alone was analyzed. Thus, treatment of DHBV-infected PDH by (D-Lys)4-PNA conjugates led to a marked inhibition of DHBV release in cell culture supernatants and to a decrease in intracellular viral replication.To better understand the role of CPPs used as a vehicle in delivery of PNAs, we examined the antiviral activity of the same anti-ε PNA but conjugated to another cationic CPP, (D-Lys)4 CPP, displayed no pronounced inhibitory activity on DHBV secretion and on intracellular DHBV DNA. Importantly, the treatment of the same cells by (D-Lys)4 CPP alone did not affect viral replication [4 and its corresponding bonding CPP-PNA conjugates displayed toxicity in PDH cells, as assessed by Neutral Red test [Interestingly, this inhibition was specific, since a 2-nucleotides mismatched PNA conjugated to the same (D-Lys)lication . RemarkaRed test .8, used as vehicles to improve cellular uptake of their bioactive PNA cargos, may reduce the sequence-specificity of CPP-PNA conjugates. In contrast, in the absence of the inhibitory effect exhibited by these cationic CPPs, the CPP-PNA conjugates are able to specifically inhibit hepadnaviral replication [Collectively, these findings demonstrate that the antiviral activity of some cationic CPPs such as (DArg)lication .Other preclinical studies demonstrated the benefit of CPP or PTD peptides used as vehicles. Indeed, PTD-p53 conjugates effectively suppressed production of HBV messenger RNAs (mRNAs), as well as HBsAg, HBeAg, and HBcAg, both in vitro and in vivo . Also, Cd-galactose-(poly)Lys- bovine serum albumin (BSA) complex improved the intracellular expression of the IX human factor gene in HepG2 cells [The asialoglycoproteins receptors (ASGP-R) were the first mammalian lectins described  and are G2 cells . MoreoveG2 cells . In addiG2 cells .In another study, PNAs hybridized to the AF/ODN conjugates were efficiently internalized into murine primary hepatocytes and in HepG2 cells through receptor-mediated endocytosis. Indeed, after a 4-h incubation, PNAs were mainly localized in the nuclei of the cells. In addition, more than 70% inhibition of telomerase activity was detected when anti-human telomerase PNA was carried to HepG2 cells using AF/ODN. An in vivo study showed that the AF/ODN-PNAs conjugates led to an effective delivery of PNAs to the liver after intravenous injection into mice .We evaluated the anti-HBV activity of sugar-modified CPP-PNA conjugates in the HepaRG human hepatoma cell line. We chose HepaRG cells for this study since these cells when differentiated share different common features with human hepatocytes, including the expression of ASGP-R on their surface. Differentiated HepaRG cells can be effectively infected with HBV, reproducing a full viral replication cycle and cccDNA formation. Moreover, HepaRG are considered as a reference in vitro cell culture model for anti-HBV drug evaluation ,63.3-PNA), could HBsAg release. We first investigated the cellular uptake of Lac-(Lys)3-PNA coupled to FITC that was incubated with uninfected HepaRG cells. Our preliminary results showed uptake of fluorescein-labeled PNA indicating that Lac-CPP-PNA conjugates can enter into the HepaRG cells RG cells .3-PNA conjugate was evaluated in HBV-infected HepaRG cells. As illustrated in 3-mismatch peptide nucleic acid (MM PNA)) showed a far less marked (3%) inhibitory effect on the release of HBsAg.Next, the antiviral effect of this Lac-(Lys)3-PNA into hepatocytes via interaction of the Lactose moiety with ASGP-R, probably through receptor-mediated endocytosis. Altogether, these data demonstrate that treatment of HBV-infected HepaRG cells by anti-S PNA coupled to Lactose increases its uptake and decreases HBsAg release, although internalization and inhibitory potential of this novel conjugate need to be further investigated.Our preliminary results suggest an uptake of Lac-(Lys)In addition to hepadnaviruses, the antiviral activity of CPPs, CPP-ODN, or CPP-protein conjugates targeting other viruses was investigated. Indeed, a monoclonal antibody targeting the HIV-1 capsid protein (p24), chemically conjugated to κFGF-MTS CPP, entered into HIV-1-infected cells and inhibited viral replication up to 73 and 49% in T-lymphocyte and in primary peripheral blood mononuclear cells (PBMCs), respectively .In another study, a modified CPP, derived from the HIV-1 TAT protein, exhibited a potent anti-HIV-1 activity in vitro. Importantly, treatment of HIV-1 infected cells with this CPP showed a direct relationship between its cationic charge and antiviral potency .Interestingly, a chimeric peptide (FP-PTD) was able to deliver specific siRNA (FP-PTD-siRNA) into HIV-1-infected cells and to efficiently inhibit viral replication .In another report, Yoo et al., showed that a CPP-PNA conjugate targeting the top of the 3′-untranslated transcribed region of massager RNA (3′-UTR) loop of the Japanese encephalitis virus (JEV) structure was effective in blocking viral proliferation .Coupling of artificial zinc-finger proteins (AZPs) to CPPs led to CPP-AZP conjugates, which appeared as pertinent antiviral agents against Human papillomavirus (HPV). Indeed, several studies demonstrated that greater internalization of CPP-AZP conjugates into mammalian cells resulted in more potent inhibition of viral replication ,69. Thus8 and Decanoyl-(D-Arg)8, alone displayed a potent antiviral effect, altering the late stages of DHBV and HBV morphogenesis. In addition, lipid and sugar domain-conjugated CPPs delivered their PNA cargos into hepatocytes and displayed an increased antiviral activity. Importantly, an anti-HBV PNA coupled to Lactose exhibited a high affinity to the asialoglycoprotein receptor on hepatocellular membranes and was shown to enter into the human hepatoma HepaRG cells and inhibit HBsAg release.Taken together, different studies demonstrated an important role of CPPs or modified-CPPs in the intracellular delivery of PNAs that target the hepadnaviral replication pathway. Indeed, in the DHBV infection model, PNA that targets the viral encapsidation ε signal and is coupled to CPPs decreased viral replication in vitro in PDHs and in vivo in ducklings. Interestingly, some CPPs, (D-Arg)Thus, these different preclinical studies provide the evidence that CPPs, modified-CPP, and CPP-PNAs conjugates represent pertinent molecular tools for future development of novel therapeutic approaches to fight chronic hepatitis B infection. Further studies are warranted to explore the antiviral potency of these compounds, targeting different steps of hepadnaviral replication, which could be used in combination with immune therapies able to break immune tolerance in HBV-carrier patients and to enhance viral infection clearance.Moreover, CPPs and modified CPPs appear of promise for development of novel antiviral strategies against different RNA and DNA viruses.


Fresh broccoli sprouts were homogenized in phosphate-buffered saline and filtered through a gauze. After centrifugation, clear extract was obtained. AGE formation was measured by enzyme-linked immunosorbent assay. Gene expression was evaluated by real-time reverse transcription-polymerase chain reaction. Reactive oxygen species (ROS) generation were measured using a fluorescent dye. Five percent broccoli sprout extract inhibited the formation of AGEs, reduced basal gene expressions of monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecule-1  and receptor for AGEs (RAGE), and upregulated endothelial nitric oxide synthase (eNOS) mRNA levels in HUVECs. TNF-α upregulated MCP-1, ICAM-1, and RAGE mRNA levels in HUVECs, all of which were attenuated by the treatment with 1% broccoli sprout extract. Pretreatment of 1% broccoli sprout extract prevented the ROS generation in HUVECs evoked by AGEs. The present study demonstrates that sulforaphane-rich broccoli sprout extract could inhibit the AGE-RAGE axis and exhibit anti-inflammatory actions in HUVECs. Supplementation of sulforaphane-rich broccoli sprout extract may play a protective role against vascular injury.We have previously shown that sulforaphane not only inhibits formation of advanced glycation end products (AGEs) but also exerts anti-inflammatory effects on AGE-exposed human umbilical vein endothelial cells (HUVECs) and AGE-injected rat aortae. Here we examined the effects of aqueous extract of glucoraphanin-rich broccoli sprouts on formation of AGEs and then investigated whether the extract could attenuate inflammatory or oxidative stress reactions in tumor necrosis factor-alpha (TNF- Sugars, such as glucose, glyceraldehyde, and methylglyoxal, can react nonenzymatically with the amino groups of lipids, proteins, and nucleic acids to form senescent macromolecules termed “advanced glycation end products (AGEs),” whose process has been shown to progress under inflammatory and hyperglycemic conditions –9. Accum in vitro and attenuates inflammatory reactions in AGE-exposed endothelial cells and AGE-infused rat aortae partly by reducing the expression of RAGE pyren-exposed Mongolian gerbils [μg quercetin/g broccoli sprout) [We found here that 5% broccoli sprout extract for 4 hours decreased basal gene expression levels of MCP-1, ICAM-1, and RAGE in HUVECs by 50-90%, while it increased eNOS mRNA levels to 1.5-fold over the control values. So, it is unlikely that broccoli sprout extract at this concentration may exert toxic effects on HUVECs. However, since the effects of 5% broccoli sprout extract on nontreated HUVECs were drastic, we chose the concentration of 1% broccoli sprout extract in the experiments of TNF-tivation . Given ttion [κB , 23, 1% nockdown . Further as well . These f gerbils . Moreove gerbils . Althoug sprout) , it is u sprout) , 27.α-treated HUVECs but also blocked the ROS generation elicited by AGEs. Therefore, broccoli sprout extract may attenuate the activation of AGE-RAGE pathway by at least two mechanisms: one is the inhibition of AGE formation and the other is the suppression of RAGE expression. Since the AGE-RAGE interaction-mediated ROS generation could further stimulate the formation of AGEs and induction of RAGE [We, along with others, have previously shown that engagement of RAGE with AGEs evokes oxidative stress generation and inflammatory reactions in HUVECs –30. In t of RAGE –33, brocAccumulating evidence has suggested that MCP-1 and ICAM-1 stimulate the recruitment and firm adhesion of inflammatory cells to endothelial cells, thereby contributing to the develop and progression of atherosclerosis –36. FurtIn this study, we could not clarify active components in broccoli sprout extract that might be responsible for the observed effects. However, the aim of the present study is to clarify whether aqueous extract of sulforaphane-rich broccoli sprout could inhibit the AGE-RAGE axis and exhibit anti-inflammatory effects in HUVECs as in the case of sulforaphane , 12. Our


Unraveling of the HLA-related immunogenetic basis of several immune disorders is complex due to the extensive HLA polymorphism and strong linkage-disequilibrium between HLA loci. A lack of in phase sequence information, a relative deficiency of high resolution genotyping including non-coding regions and ambiguous haplotype assignment make it difficult to compare findings across association studies and to attribute a causal role to specific HLA alleles/haplotypes in disease susceptibility and modification of disease phenotypes. Earlier, historical antibody and DNA-based methods of HLA typing, primarily of low resolution at antigen/alellic group levels, yielded “indicative” findings which were partially improved by high-resolution DNA-based typing. Only recently, next-generation sequencing (NGS) approaches based on deep-sequencing of the complete HLA genes combined with bioinformatics tools began to provide the access to complete information at an allelic level. Analyzing HLA with NGS approaches, therefore, promises to provide further insight in the etiopathogenesis of several immune disorders in which HLA associations have been implicated. These range from coeliac disease and rheumatological conditions to even more complex disorders, such as type-1 diabetes, systemic lupus erythematosus and sarcoidosis. A systemic disease of unknown etiology, sarcoidosis has previously been associated with numerous HLA variants and also other gene polymorphisms, often in linkage with the HLA region. To date, the biological significance of these associations has only partially been defined. Therefore, more precise assignments of HLA alleles/haplotypes using NGS approaches could help to elucidate the exact role of HLA variation in the multifaceted etiopathogenesis of sarcoidosis, including epigenetic mechanisms. NGS-based HLA analyses may be also relevant for defining variable clinical phenotypes and for predicting the disease course or the response to current/plausible novel therapies. HLA gene expression is further activated by cytokines  produced during both innate and adaptive immune responses. The HLA region has been associated with numerous diseases, primarily, of an autoimmune or infectious nature  region, a 3.6 Mb segment of the human genome at 6p21.3, encodes more than 220 genes of diverse function, including 20 immune protein-coding genes. The codominant expression of HLA genes regulates the acquired immune response as HLA-class I and HLA-class II genes interact with T cell receptors (TCRs) of CD8s nature .HLA-A, -B, and -C and class II HLA-DRB1, -DQB1, and -DPB1 (killer-cell Ig-like receptors ‘KIR’) and by cells of monocyte lineages (leukocyte Ig-like receptor ‘LIR’)  . Thus, H∗IMP:02, and HLA-check) to infer the classical HLA alleles and amino acid changes using the LD information existing between HLA alleles and SNP markers in the HLA region (∗08:01HLA-B rs4143332) (PCR-based HLA typing using sequence-specific primers (SSP), sequence-specific oligonucleotide probes (SSOP), and Sanger sequencing-based typing (SBT) methods have significantly improved HLA typing resolution, however, these methods possess several limitations, including time-consuming protocols, low throughput, unphased data and ambiguity . Single A region . HoweverA region  remains A region . The nom4143332)  may be hHLA polymorphism was first identified and characterized using alloantibodies (antisera) against leukocytes and the microlymphocytotoxicity test was developed for serological typing of HLA antigens (tissue types) in humans . HoweverNGS platforms commonly in use for high-throughput sequencing and massively parallel analysis, including for HLA encompass Ion torrent (Thermo Fisher), Illumina, SOLiD (ABI), PacBio, Oxford Nanopore. In addition to capital costs, they differ in various features, including sequence length and sequencing time; see Supplementary Table For NGS, the two main clonal techniques specifically targeting the HLA region include, (i) long/mid-range PCR based isolation, and (ii) hybridization-based capture methods; however, achievement of good allelic balance and homogenous coverage along all the target genes remains a major challenge . The com1′ (2 (CWD v2.0.0)  . The CWDB1 genes .HLA-DPB1 and BTNL2 genes and five loci among HLA-C, -B, -DPB1, and BTNL2 were reported using NGS , infectious diseases  and other conditions such as malignancies and neuropathies . In mostsing NGS . Distincsing NGS .In future, allelic resolution of HLA genes obtained by NGS will be helpful in studying the effect of variant interactions in disease risk, in selecting the participants for prevention or intervention trials  and in d∗08:01, -B∗08:04, -B∗13:01, and -B∗13:02 . These gene variants have been shown to associate with sarcoidosis phenotypes and sarcoidosis disease course (C6orf10∗rs3129927 and HLA-DRA∗rs3135394 was reported for Löfgren’s syndrome (LS) – a particular phenotype of more benign disease. By contrast, BTNL2∗rs2076530 and HLA-DRA∗rs3129882 was associated with the non-LS sarcoidosis  were reported with improved resolution using NGS HLA typing  as increased or decreased risk variants in disease phenotypes and disease progression in sarcoidosis has been outlined (HLA-DQA and -DQB genes and about 96% of European coeliac patients carry the HLA-DQ2.5 heterodimer (∗05DQA1-∗0201DQB1)   and in sB1∗0201) . Also, tulations .allelefrequency.net  with the promoter region of HLA-F (HLA-DQB1 gene in tuberculosis patients was associated with reduced expression of HLA-class II genes (HLA-DRA gene expression through multiple complexes of histone lysine acetyltransferase (KAT) and histone lysine methyltransferase (KMT) respectively, in a class II transactivator (CIITA) independent or dependent manner . In the context of HLA, non-coding sequences from HLA regions, particularly the regulatory enhancers, promoter and untranslated regions (5’- and 3’-UTRs) could form a repertoire for non-genetic factors acting as epigenetic regulators of HLA genes. Primarily, the regulatory elements have differential CpG methylation and miRNA binding sites, while histone modifications are derived by specific enzymes or protein complexes. Interestingly, HLA was among the first regions explored for methylation profiling, the HLA region can be analyzed for both genetic and epigenetic features . The evipression . Methylaof HLA-F . FurtherII genes . Histonet manner . The UTRilencing  and HLA-n asthma .HLA-A 3’-UTR (100 bp insertion) affecting RNA binding protein Syncrip was shown to modulate the post-transcriptional expression of the HLA-A protein  in the seed domain, which eventually modulates the target gene expression . Similar protein . A concegulation . Secondlpathways . It is cquencing .HLA typing has already benefited and will continue to benefit from NGS applications. Comprehensive and unambiguous characterisation of extensive polymorphisms in the HLA region by NGS will contribute to progress in the area of HLA and disease associations by precise identification of these associations at the allelic level and by utilizing exact data from NGS-based population genetic studies. In sarcoidosis, the translation of expected developments to clinical practice will aim at defining new genetic markers for particular disease phenotypes and at using knowledge on the nature of yet unknown peptides presented by HLA molecules for understanding disease mechanisms, resulting in development of specifically tailored therapies. In our opinion, dynamic developments in NGS methodologies and linked bioinformatic analyses will ultimately expand clinical applications of HLA typing beyond traditional areas of transplantation and immunogenetics. Understanding the molecular mechanisms of sarcoidosis using these novel approaches represents just a small piece at the start of these endeavors.MP and AK contributed to the conception and writing of this review. Both authors have reviewed and approved the submitted work.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.


Drosophila imaginal discs. For the Hox TF Antennapedia, we investigated whether protein variability results from random stochastic events or is developmentally regulated. We found that Antennapedia transitioned from low concentration/high variability early, to high concentration/low variability later, in development. FCS and temporally resolved genetic studies uncovered that Antennapedia itself is necessary and sufficient to drive a developmental regulatory switch from auto-activation to auto-repression, thereby reducing variability. This switch is controlled by progressive changes in relative concentrations of preferentially activating and repressing Antennapedia isoforms, which bind chromatin with different affinities. Mathematical modeling demonstrated that the experimentally supported auto-regulatory circuit can explain the increase of Antennapedia concentration and suppression of variability over time.The variability in transcription factor concentration among cells is an important developmental determinant, yet how variability is controlled remains poorly understood. Studies of variability have focused predominantly on monitoring mRNA production noise. Little information exists about transcription factor protein variability, as this requires the use of quantitative methods with single-molecule sensitivity. Using Fluorescence Correlation Spectroscopy (FCS), we have characterized the concentration and variability of 14 endogenously tagged TFs in live  Summary: Preferentially repressing and activating isoforms of the Hox transcription factor Antennapedia elicit a developmental regulatory switch from auto-activation to auto-repression that increases concentration and suppresses cell-to-cell variability over time. In order to understand the mechanisms that control pattern formation and cell fate specification in developing organisms, the intranuclear concentration, DNA-binding kinetics and cell-to-cell variability of relevant transcription factors (TFs) need to be quantified. TF concentration variability at the tissue level is thought to arise from diverse processes, including mRNA transcription, translation and protein degradation. Intrinsic noise is due to stochastic binding and interactions of proteins involved in transcriptional activation of a specific gene . ExtrinsIn undifferentiated tissue or cells, TF cell-to-cell variability can be the driving force for differentiation. For example, progressive establishment of a Nanog salt-and-pepper expression pattern leads to the formation of primitive endoderm in the mouse preimplantation embryo, whereas loss of the variability results in embryos lacking primitive endoderm entirely .Drosophila gastrulation  TF, which is required for the invagination of the mesoderm during rulation , or the rulation .In addition, differential cell fates within the same developmental territory may be specified by TFs deploying different DNA-binding dynamics, despite the existence of very similar concentrations (i.e. low variability). For example, studies on the Oct4 TF in early mouse embryos have shown that differential kinetic behavior of DNA binding, despite equal Oct4 concentration among blastomeres, ultimately dictates an early developmental bias towards lineage segregation .So far, studies of gene expression variability have focused predominantly on monitoring the noise of mRNA production . Little in vivo, proteins need to be quantified at endogenous levels (We have previously used Fluorescence Correlation Spectroscopy (FCS) to quantitatively characterize Hox TF interactions with chromatin in living salivary gland cells . FCS is s levels .in vivo by FCS, and their cell-to-cell variability in fly imaginal discs. Imaginal discs are flat, single-layered epithelia comprising small diploid cells and many TFs are expressed in defined regions within these tissues during development.In this study, we take advantage of the availability of fly toolkits, in which TFs have been endogenously tagged using different methodologies, fosmid , BAC de and MiMIFig. S1A-P). FCS is a non-invasive method with single-molecule sensitivity, in which a confocal arrangement of optical elements is used to generate a small (sub-femtoliter) detection volume inside living cells, from which fluorescence is being detected , information can be obtained regarding: (1) the absolute TF concentrations  TFtrations G; and . Various diffusion times and fractions of slow- and fast-diffusing TF molecules .We selected 14 TFs based on the availability of homozygous endogenously tagged transgenes and on the generation of robust fluorescence in distinct patterns in various imaginal discs. For the 14 TFs, we measured average concentrations ranging across about two orders of magnitude among the different TFs, from ∼30 nM to ∼1.1 μM  , in line with Fano factor values of other TFs determined previously to lie between 0 and 30  in high average concentrations, in particular in the leg disc , we performed this analysis in the wing disc where expression levels are lower than in the leg disc . FCS showed that different clusters of neighboring cells along the Antp expression domain in the wing disc display different average expression levels . Such behavior is indicative of complex transcriptional regulatory processes  transgenes fused to eGFP (SynthAntp-eGFP). These transgenes encode the Antp protein (amino acids 278-378), which includes the homeodomain, the conserved YPWM motif and the C terminus  and they harbor Antp-specific homeotic function  repressed the endogenous Antp protein, indicating that Antp is indeed able to regulate its own protein levels.One mechanism by which genes control their expression level variability is auto-regulation . To testfunction . Clonal Antp locus is subject to complex transcriptional regulation, involving a distal and a proximal promoter , spanning more than 100 kb of regulatory sequences. We established that the P1 promoter (rather than the P2 promoter) is predominantly required to drive expression of Antp in the wing disc notum , in line with previous observations . To rule out putative dominant-negative activity of the small SynthAntp-eGFP peptide, we also performed these experiments with the full-length Antp protein  and found them to also repress the reporter. We conclude that the Antp protein is able to repress its own transcription from the P1 promoter (directly or indirectly), suggesting a possible mechanism of suppressing cell-to-cell variability of Antp expression levels  (MD23) enhancer resulted in activation of the Antp P1 reporter in distal compartments of the wing disc, such as the wing pouch, where Antp is normally not detected . This suggests that as well as its auto-repressing function, Antp is also capable of activating its own transcription . We observed indistinguishable DNA-binding behavior by FCS, not only across the whole concentration range examined , but also between endogenous and overexpressed Antp . Importantly, the auto-activating and auto-repressing capacity of Antp was preserved even with the weak Gal4-driver 69B  that triggered concentrations of Antp lower than its normal concentration in the leg disc (473 nM versus 1110 nM), indicating that auto-activation and auto-repression of Antp take place at endogenous protein concentrations. We conclude that Antp is able to repress and activate its own transcription  or at 60 h  of development and analyzed the clones in late third instar wing imaginal discs . Clonal induction of full-length untagged Antp in early development triggered strong auto-activation of Antp-eGFP . As before, we confirmed that early auto-activation of Antp is transcriptional and similar for both full-length and SynthAntp proteins . Early auto-activation was further supported by results from a loss-of-function experiment, where RNAi-mediated early knockdown of Antp resulted in downregulation of the Antp reporter . The loss- and gain-of-function analysis together suggest that during early disc development Antp is required for sustaining its own expression.To further investigate the mechanism by which the al discs . We chosal discs . As a prIn contrast, clonal induction during the late second instar stage G repressTogether, these results revealed the existence of a switch in Antp auto-regulatory capacity on its own transcription during development. Starting from a preferentially auto-activating state early in development D, Antp cAntp auto-repressive state limits the variability of Antp protein concentration among neighboring cells late in development, we expected that the variability would be higher during earlier stages, when auto-repression does not operate. We therefore used FCS to characterize the endogenous expression levels and cell-to-cell variability of Antp concentration in nuclei of second instar wing and leg discs. We observed significantly lower average concentrations of Antp protein in second versus third instar wing and leg discs, and the inverse was true for concentration variability , indicating that the developmental increase in concentration is accompanied by suppression of concentration variability. In addition, FCS revealed a notable change in Antp characteristic decay times  at early versus late stages . This behavior indicates that endogenous Antp is initially moving fast in the nucleus, as it undergoes considerably fewer interactions with chromatin compared with later stages where its interactions with chromatin are more frequent and longer lasting.If the Antp is expressed at relatively low and highly variable levels in early developing discs, when genetic evidence indicates auto-activation capacity on its own transcription. Later in development, when Antp has reached a state of higher average concentrations, auto-repression kicks in, resulting in considerably lower variability among neighboring cells.Taken together, our measurements show that Antp auto-activation and repression. It is well established that the Antp mRNA contains an alternative splice site in exon 7 immediately upstream of the homeobox-containing exon 8, and generates Antp isoforms differing in as little as four amino acids in the linker between the YPWM motif (a co-factor-interacting motif) and the homeodomain . Inversely, long-linker Antp isoforms exhibited stronger activation of Antp reporter, when compared with short-linker isoforms . We conclude that, also in the case of Antp auto-regulation, short-linker isoforms function as more potent repressors, whereas long-linker isoforms operate as more potent activators.Ectopic expression of SynthAntp-eGFP peptides featuring a long linker displayed significantly weaker repression capacity on endogenous Antp, when compared with their short linker counterparts  endogenous levels . FCS measurements revealed that the short-linker isoform displayed longer characteristic decay times and a higher fraction of DNA-bound molecules, suggesting stronger and more pronounced binding to chromatin than its long-linker counterpart . With chromatin (and therefore Antp-binding sites configuration), as well as the presence of co-factor proteins, being identical between the two instances , we were able to directly compare the apparent equilibrium dissociation constants for the two isoforms . We found that the affinity of binding to chromatin . To corroborate these findings, we also performed gel-shift experiments to test how full-length recombinant Antp isoforms, which bear a short and a long linker, bind previously characterized Antp-binding sites. We found that equal amounts of Antp long-linker isoform bind Antp-binding sites more weakly than its short linker counterpart . Collectively, these experiments support the notion that differences in Antp regulation during disc development can be largely attributed to differences in the affinity of the investigated Antp isoforms.Relative changes in Antp transcript variant abundance D,H, diffntp gene B-D,F-H, pression  and the pression E all poiTaken together, the switch of Antp from an auto-activating to an auto-repressing state and the alteration of its DNA-binding behavior during disc development can be largely explained by a temporal developmental regulation of the relative concentrations of preferentially auto-activating and auto-repressing Antp protein isoforms. These isoforms display distinct properties in their modes of interaction with chromatin E,I.SynthAntp ectopically in the antennal disc, which is devoid of endogenous Antp expression, and monitored the extent (strength) of homeotic transformations induced by different Gal4 drivers corresponding to different SynthAntp concentrations . In this experiment, expression of SynthAntp is controlled by the Gal4 driver, independently of the Antp locus, therefore the phenotypic output does not depend on Antp auto-regulation. We observed that partial transformations of antennae to tarsi could be obtained with drivers expressing Antp at close to endogenous concentration . Therefore, Antp can repress the antennal and launch the leg developmental program in the antennal disc at endogenous concentrations, although not robustly across the tissue . As expected, the three weak transformation phenotypes, elicited by ptc-, Dll (MD713)- and 69B-Gal4  enhancer resulted in robust homeotic transformation to a complete tarsus (CV2=0.103). Importantly, endogenous Antp and Antp overexpressed by any of the Gal4 drivers showed indistinguishable chromatin-binding behavior by FCS . Therefore, robust Antp homeotic function can be achieved at concentrations that are accompanied by low variability.The mechanism of developmental Antp auto-regulation offered a possible explanation for the observed increase in Antp concentration from second to third instar discs, as well as the suppression of variability. An unresolved issue is the functional significance of suppression of Antp variability in development. To test this, we need to manipulate variability, yet this is currently not possible to achieve at the endogenous locus. However, as average concentration and variability are interdependent, we used an ectopic expression system to progressively dampen Antp variability by manipulating its concentration. To achieve this, we expressed 69B-Gal4 B-D, were69B-Gal4 E,F. In ce tarsus A, accompe tarsus F. This cAntp expression . This model tests whether positive and negative auto-regulation of Antp through distinct isoforms is sufficient to explain the increase in protein concentration and decrease in nucleus-to-nucleus variability from early to late stages. The model consists of a dynamic promoter, which drives transcription of Antp followed by a splicing step, yielding either the auto-repressing or the auto-activating isoform of Antp. As the repressing isoform has higher abundance at later stages, we assumed that splicing is more likely to generate this isoform than the activating isoform. The initial imbalance of Antp towards the activating isoform . With extrinsic factors affecting both genes similarly, we expected a correlation between the concentration of nuclear mRFP1 and Antp-eGFP. Our data showed a statistically significant correlation between mRFP1 and Antp . Correspondingly, we accounted for extrinsic variability also in our model by allowing gene expression rates to randomly vary between cells . We analyzed both model variants by forward simulation and found that both of them can explain the switch-like increase in average Antp concentration between early and late stages  and the relative fraction of repressing and activating isoforms . However, only the non-competitive binding model . Simulation trajectories of individual nuclei indicated an initial increase and a subsequent stabilization of concentration, whereas in the competitive model, or in the absence of the negative feedback, this is not achieved . Additionally, we established that the negative feedback is required for suppression of variability , as otherwise no suppression of variability is conferred . Thus, the model suggested that auto-repression is required and that isoforms do not compete for binding to the P1 promoter.In this promoter model, it remains unclear whether the two isoforms compete for the same binding sites on the P1 promoter. In this case, an increase in concentration of repressing Antp species enhances the probability to reach state ‘R’ only if the promoter is in state ‘U’ G. In theng model G can expTo further validate this model, we analyzed how Antp variability scales with average concentrations, compared with our experimental measurements. To generate different average concentrations, we varied the gene expression rates over three orders of magnitude. The model predicted a decrease in variability as a function of total Antp concentration and an increase in the Fano factor. These findings are in good agreement with the experimental data (compare SynthAntp transgenes (bearing either a long or a short linker isoform) from the Antp P1 promoter  resulted in 100% embryonic lethality, rendering the analysis of concentration and variability in imaginal discs impossible. This indicated that an indiscriminate increase of the dose of either Antp variant from early embryonic development onwards cannot be tolerated or buffered by the auto-regulatory circuit.We next analyzed the model behavior under different genetic perturbations. Increase of Antp concentration by overexpressing Dll enhancer [Dll-Gal4 (MD23)] in the leg discs or in the notum , which overlaps with the endogenous Antp expression pattern only during first instar disc development  displayed the wild-type morphology, indicative of normal Antp function, regardless of which isoform (activating or repressing) was overexpressed. We further measured by FCS the concentration and variability of the total Antp protein (endogenous Antp-eGFP and overexpressed SynthAntp-eGFP) in proximal regions of the leg disc at second and third instar stages , resulted in abnormal leg  development. These flies died as pharate adults with malformed legs, in line with Antp being required for proper leg development in all ventral thoracic discs (legs). FCS measurements in the corresponding proximal leg disc cell nuclei of second and third instar leg discs overexpressing mCherry-SynthScr revealed pronounced reduction in Antp concentration and remarkable increase in variability , which can repress Antp at the transcriptional level, but can neither activate it nor activate its own transcription  in haltere specification and as a mechanism of controlling Ubx levels against genetic variation (Ubx promoter regulation in Drosophila S2 cells (Deformed expression in the embryo (Negative auto-regulation has been identified as a frequently deployed mechanism for the reduction of noise (cell-to-cell variability) and the increase of regulatory robustness in various systems . Auto-reariation , as wellS2 cells . In conte embryo . These ein vivo assays. Finally, the Ubx linker also affects the strength of its interaction with the Hox co-factor Extradenticle (Exd), underscoring the functional importance of linker length in Hox TF function . This ‘buffering’ capacity on cell-to-cell variability is reflected in the ability of flies to tolerate more than threefold overexpression of Antp without dramatic changes in endogenous Antp levels or generation of abnormal phenotypes. Therefore, two different isoforms produced from the same gene with opposing roles in transcriptional regulation and different auto-regulatory binding sites on the promoter of the gene seem to suffice to create a robust gene expression circuit that is able to ‘buffer’ perturbations of the starting conditions.Mathematical modeling predicts that the So far, we have only been able to indiscriminately increase or decrease Antp concentration at the tissue level and record the phenotypic outcome of these boundary states. It will be interesting to test whether controlled perturbations of TF variability at the tissue level that render TF concentration patterns less, or more, noisy among neighboring cells, while maintaining similar mean protein concentrations, lead to abnormal phenotypes. The technology to selectively manipulate expression variability of specific TFs in a developing tissue is yet to be established.atonal (VDRC ID 318959), brinker (VDRC ID 318246), spalt major (VDRC ID 318068), yorkie (VDRC ID 318237), senseless (VDRC ID 318017) and Sex combs reduced (VDRC ID 318441) fosmid lines are available from the Vienna Drosophila Resource Center (VDRC) and were generated recently in our laboratory (fork head (stock 43951), grainy head (stock 42272), Abdominal B (stock 38625), eyeless, (stock 42271), spineless  and grain (stock 58483) tagged BACs were generated by Rebecca Spokony and Kevin P. White  and are available at the Bloomington Stock Center. For the scalloped gene, a GFP-trap line was used (ey13630::2XTY1-SGFP-V5-preTEV-BLRP-3XFLAG)dFRT line (VDRC ID 318018) has been tried also in fluorescence imaging and FCS experiments, but did not yield detectable fluorescence in the eye disc for it to be used in our analysis. The act5C-FRT-yellow-FRT-Gal4  line used for clonal overexpression or RNAi knockdown has been described (Antp lines (synthetic and full-length), as well as UAS-SynthScr constructs, have been previously described  line was a kind gift from Ginés Morata  . We are indebted to Sebastian Dunst  for generating the ubi-FRT-mCherry(stop)-FRT-Gal4(VK37)/CyO line, which drives clonal overexpression upon flippase excision, while simultaneously marking cells by the loss of mCherry. For red-color labeling of clones the act5C-FRT-CD2-FRT-Gal4, UAS-mRFP1 (NLS)/TM3 stock 30558 from the Bloomington Stock Center was used. For marking the ectopic expression domain of untagged Antp proteins, the UAS-mRFP1(NLS)/TM3 stock 31417 from the Bloomington Stock Center was used. The MS243-Gal4; UAS-GFP/CyO line was a kind gift from the laboratory of Ernesto Sánchez-Herrero .The Antp-eGFP MiMIC line was a kind gift from Hugo J. Bellen . The boratory . The forwas used , a kind was used . For theescribed . The UASescribed , 2010. T, Spain) . 69B-Galescribed . The P1 hs-flp/+; act5C-FRT-yellow-FRT-Gal4/+; UAS-SynthAntp long linker-eGFP/+hs-flp/+; act5C-FRT-yellow-FRT-Gal4, UAS-eGFP/+; UAS-Antp long linker /+Dll-Gal4 (MD23)/+; UAS-SynthAntp-eGFP/Antp P1-lacZhs-flp/+; ubi-FRT-mChery-FRT-Gal4/+; Antp-eGFP (MiMIC)/UAS-Antp long linker hs-flp/+; UAS-AntpRNAi/+; Antp P1-lacZ/act5C-FRT-CD2-FRT-Gal4, UAS-mRFP1(NLS)hs-flp/+; UAS-AntpRNAi/act5C-FRT-yellow-FRT-Gal4, UAS-eGFP; Antp P1-lacZ/+ptc-Gal4/+; UAS-SynthAntp long linker-eGFP/+Dll-Gal4 (MD23)/+; UAS-SynthAntp long linker-eGFP/Antp P1-lacZptc-Gal4/+; UAS-SynthAntp long linker-eGFP/+Dll-Gal4 (MD23)/+; UAS-SynthAntp short linker-eGFP/Antp P1-lacZDll-Gal4 (MD23)/+; UAS-SynthAntp long linker-eGFP/+ptc-Gal4/+; UAS-SynthAntp long linker-eGFP/+Dll-Gal4 (MD713)/+; UAS-SynthAntp long linker-eGFP/+69B-Gal4/UAS-SynthAntp long linker-eGFPDll-Gal4 (MD23)/+; UAS-SynthAntp long linker-eGFP/+ or Dll-Gal4 (MD23)/+; UAS-SynthAntp short linker-eGFP/+Dll-Gal4 (MD23)/+; UAS-mCitrine-SynthScr/+For FCS measurements, imaginal discs  and salivary glands were dissected from third instar wandering larvae, or wing and leg discs from second instar larvae, in Grace's insect tissue culture medium  and transferred to an eight-well chambered coverglass  containing PBS just prior to imaging or FCS measurements. Floating imaginal discs or salivary glands were sunk to the bottom of the well using forceps.Antp P1 promoter staining in imaginal discs, we used the mouse anti-β-galactosidase 40-1a antibody from Developmental Studies Hybridoma Bank, University of Iowa at 1:50. The rabbit anti-Scr antibody was used at 1:300  and larval expression of Antp and eGFP. In this experiment, we (1) visualized the overlap between eGFP and Antp ; and (2) compared the eGFP expression pattern to the Antp expression pattern in wild-type discs and embryos.To examine whether the pattern of the MiMIC Antp-eGFP fusion protein recapitulates the deformed (dfd)-YFP bearing balancer chromosomes . For detection of the long linker variant, we designed primers either corresponding to the RSQFGKCQ sequence or to GKCQERKR. We observed in control PCRs (using plasmid DNA harboring either a long or a short linker cDNA) that primers designed for the short linker variant still amplified the long linker one. Moreover, with linker sequences differing in only four amino acids, encoded by 12 base pairs, primer pairs flanking the linker could also not be used, because, owing to very similar sizes, both variants would be amplified in RT-qPCR experiments with almost equal efficiencies. Therefore, we used primer pairs flanking the linker region to indiscriminately amplify short and long linker variants, using non-saturating PCR (18 cycles) on total cDNA generated from total RNA. We then resolved and assessed the relative amounts of long and short linker amplicons in a second step using Fragment Analyzer . RNA was extracted from stage 13 embryos, second instar larvae at 60 h of development, and leg or wing discs from third instar wandering larvae using the Trizol reagent (Thermo Fisher Scientific), following the manufacturer's instructions. Total RNA amounts were measured using NanoDrop and equal amounts were used to synthesize cDNA using the High-Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific), following the manufacturer's instructions. Total cDNA yields were measured by NanoDrop and equal amounts were used in PCR, using in-house produced Taq polymerase. Plasmid DNA (10 ng), bearing either a long or a short transcript cDNA were used as a control. PCR product abundance was analyzed both by agarose gel electrophoresis and using Fragment Analyzer.y-axis) as the sum of long and short isoforms at each developmental stage, whereas the quantification of the relative activation and repression efficiency was performed considering the short linker variant as having 100% repression and the long linker variant as having 100% activation efficiency .The quantification of the transcript variant concentration D,H was mQuantification of the relative efficiency of Antp activating and repressing isoforms D,H were –Marquardt algorithm. Quality of the fitting was evaluated by visual inspection and by residuals analysis. Control FCS measurements to assess the detection volume were routinely performed prior to data acquisition, using dilute solutions of known concentration of Rhodamine 6G and Alexa488 dyes. The variability between independent measurements reflects variabilities between cells, rather than imprecision of FCS measurements. For more details on fluorescence microscopy imaging and FCS, refer to the supplementary Materials and Methods.Fluorescence imaging and FCS measurements were performed on two uniquely modified confocal laser scanning microscopy systems, both featuring the ConfoCor3 system (Zeiss) and consisting of either an inverted microscope for transmitted light and epifluorescence (Axiovert 200 M); a VIS-laser module comprising the Ar/ArKr , HeNe 543 nm and HeNe 633 nm lasers and the scanning module LSM510 META; or a Zeiss LSM780 inverted setup, comprising Diode 405 nm, Ar multiline 458, 488 and 514 nm, DPSS 561 nm and HeNe 633 nm lasers. Both instruments were modified to enable detection using silicon Avalanche Photo Detectors  for imaging and FCS . Images supplementary Materials and Methods), yield information about the absolute concentration of fluorescent molecules , seven to ten larvae of each fly strain were dissected, yielding at least 15 imaginal discs, which were used in FCS analysis. For the Fkh TF, seven pairs of salivary glands were analyzed and for AbdB, 12 genital discs were dissected from 12 larvae. More than 50 FCS measurements were performed in patches of neighboring cells of these dissected discs, in the regions of expression indicated in Fig. S1 by arrows. Imaginal discs from the same fly strain (expressing a given endogenously tagged TF) were analyzed on at least three independent occasions (FCS sessions), taking place on different days . For Antp, which was further analyzed in this study, more than 20 independent FCS sessions were used. As routinely carried out with FCS measurements in live cells, these measurements were evaluated during acquisition and subsequent analysis, and, based on their quality (high counts per molecule and low photobleaching), were included in the calculation of concentration and variability. In Fig. S1Q, n denotes the number of FCS measurements included in the calculations.For the measurement of TF molecular numbers and variability  = 1.36×10−15 and P (repression clone vs surrounding Antp reporter) = 3.17×10−16].t-test .t-test {***P<0.001 and *P<0.05, namely P (early activation clone vs surrounding Antp protein) = 6.23×10−13 and P [early knockdown (RNAi) clone vs surrounding Antp reporter] = 2.98×10−9}.t-test .t-test {***P<0.001 and *P<0.05, namely P (late repression clone vs surrounding Antp protein) = 3.98×10−17 and P [late knockdown (RNAi) clone vs surrounding Antp reporter] = 1.16×10−21}.t-test between measurements performed with the long linker (auto-activating) isoform  = 3.16×10−5, P (2nd instar long vs short concentration) = 1.16×10−4, P (long vs short relative activation) = 4.1×10−3, P (long vs short relative activation) = 4.1×10−3, P (long vs short relative repression) = 2.4×10−4 and P [long vs short DNA-bound fraction (FCS)] = 5.6×10−10}. isoform D and the isoform H {***P<0t-test .t-test P  = 0.679 and P  = 0.454.t-test P  = 0.892.


Risk-taking is a complex form of decision-making that involves calculated assessments of potential costs and rewards that may be immediate or delayed. Thus, making predictions about inter-individual variation in risk-taking due to personality traits, decision styles or other attributes can be difficult. The association of risk-taking with gender is well-supported; males report higher propensity for risk-taking and show higher risk-taking on tasks measuring actual risk-taking behavior. Risk-taking also appears to be associated with circadian phenotypes (chronotypes), with evening-types reporting higher levels of risk-taking—but this association may be confounded by the fact that, in certain age groups, males are more likely to be evening-types. Here, we test for gender by chronotype effects on risk-taking in young adults (n = 610) using a self-reported risk propensity questionnaire, the health domain of the DOSPERT, and a behavioral task measuring risk-taking, the Balloon Analog Risk Task (BART). Our results show that males report and take significantly more risks than females in this population. In addition, evening-type individuals have significantly higher self-reported risk propensity and tend to take more risks on the BART. Interestingly, there is no significant difference in risk propensity or risk-taking behavior across male circadian phenotypes, but evening-type females significantly report and take more risk than female intermediate and morning types. In regression analyses, we found both gender and chronotype predict risk propensity and risk-taking. Path analysis confirms that chronotype has an indirect effect on gender differences in both risk propensity and risk-taking. Furthermore, we found that trait anxiety (STAI) and sleep disturbance (PROMIS), significantly correlate with chronotype and gender in the complete dataset, but do not independently predict differences in female risk-taking. These results suggest that chronotype mediates gender effects on risk-taking and that these effects are driven primarily by morning-type females, but are not related to gender-specific differences in trait anxiety or sleep quality. Individuals vary in the timing of internal circadian rhythms, resulting in variation in sleep-wake cycles (sleep-wake chronotypes) and/or preferences for timing of activities  –5. TheseCircadian phenotypes are associated with shifts in both molecular and physiological timing. The timing of individual circadian timing likely interacts with physiological pathways regulating energetic resources that influence cognitive processes, including self-control mechanisms –15. ThesRisk-taking differs from other types of cognitive decision-making in that it involves an evaluation of potential rewards and costs, which can vary with context. Thus, risk-taking has been viewed as both a stable trait (propensity to take risks) and a state  . RelativLimited previous research on circadian effects on risky decision-making suggests that chronotype affects both the propensity to take risks and risk-taking behavior in humans. Killgore (2007) split a sample of 54 young adults  into evening-types and morning types based on self-reported sleep patterns and found that evening types have a greater propensity for risk-taking on the Evaluation of Risks Scale, but did not take more risks on the Balloon Analog Risk Task (BART) . It is nAlthough there are still gaps in our understanding of how gender moderates behavior, studies have shown that personality traits, like impulsivity, and their influence on behavior, can differ between genders –33. ThusIn the current study, we test for gender and chronotype effects on risk-taking in a sample of young adults (n = 610), including undergraduate and graduate school students. We use an established risk propensity measure that is relevant to this particular population , and a well-supported risk-taking task, the Balloon Analog Risk Task (BART). We characterize the circadian phenotypes of our participants with the full, self-reported, Horne-Ostberg Morningness-Eveningness questionnaire (HO-MEQ) and include intermediate chronotypes in our analysis. We test whether risk-propensity and risk-taking are associated with both chronotype and gender, and explore whether the relationship between chronotype and risk differs between genders.Undergraduate students at Colgate University and graduate students at Cornell SC Johnson College of Business volunteered to participate in the study  yrs; range 17–58). All methods adhered to the principles of the Declaration of Helsinki; the Institutional Review Board at Colgate University and Cornell University approved all procedures and consent forms . All participants gave written informed consent. The requirement of parental consent for involvement of university students under the age of 18 in this study was waived by the Institutional Review Board at Colgate University.Each participant took a computer-based survey with questions from the full (19-question) Horne-Östberg Morningness-Eveningness Questionnaire (MEQ), the trait version of the Spielberger’s State-Trait Anxiety Scale (STAI), the short form of the Patient-Reported Outcomes Measurement Information System (PROMIS) Sleep Disturbance and the health questionnaire portion of the DOSPERT, and then completed a Balloon Analog Risk Task (BART) ,29,37–39Horne-Östberg MEQ scores range from 30 (extreme morning preference) to 70 (extreme evening preference). Individuals were classified as follows: MEQ scores ≤41 were designated evening-types (ET), MEQ ≥ 59 were designated morning types (MT), and between 42–58 were intermediate types (IT).The Domain-Specific Risk Attitude Scale (DOSPERT) was used to measure participants’ propensity of risk-taking behavior . This suActual risk-taking behavior was measured using the Balloon Analog Risk Task (BART), a computerized measure of risk-taking behavior . ParticiSleepiness is known to influence metabolic physiology and may modulate decision-making independent of circadian effects. Participants completed the short form PROMIS sleep disturbance questionnaire, allowing us to compare measures of risky decision-making with a standardized measure of sleep disturbance . Scores We measured Spielberger Trait Anxiety Scale (T-Anxiety from the STAI) which evaluates aspects of “anxiety proneness,” including general states of nervousness, worry, and tension .Differences between diurnal preference chronotypes  and genders in risk propensity (DOSPERT scores) and behavioral risk-taking (BART scores) were tested with GLM ANOVAs. Post-hoc analyses were tested with Tukey’s post-hoc tests.To investigate the relationships among the study variables, bivariate correlations were computed for Pearson’s coefficients. A hierarchical linear regression analysis was performed to test how gender, chronotype, trait anxiety, and sleep disturbance predicted risk propensity and/or risk-taking. Model 1 tested how gender predicts risk-taking, Model 2 tested the effects of gender and chronotype, Model 3 tested the effects of gender, chronotype and trait anxiety, and Model 4 included the final study variable, sleep disturbance. We performed two mediation analyses using the PROCESS macro ,41 that Risk propensity was significantly different across diurnal preference groups  = 11.355, p<0.001, Risk propensity was greater in males compared to females  = 5.160, p = 0.023; Overall, males took significantly more risks on the BART than females  = 17.631, p<0.001; gender = -0.168, p<0.001); rchronotype = -0.205, p<0.001; gender = -0.232, p<0.001); rchronotype = -0.108, p = 0.005; gender = -0.131, p = 0.001); rchronotype = -0.212, p<0.001) and sleep disturbance ; rchronotype = -0.467, p<0.001).Results of correlation analysis showed that both gender and chronotype were significantly and negatively correlated with risk propensity  and 5.4% of the variance explained by gender for risk-taking on the BART . The asOne of the leading hypotheses for high risk-taking in evening-types involves physiological processes related to circadian rhythms and social jetlag ,30. SociGender differences in anxiety may also influence risk-taking; females tend to have higher levels of anxiety, and this population shows significantly higher anxiety levels in females relative to males. State anxiety has been shown to modulate gender differences in risk-taking  and traiThe strengths of this study include a large sample size and a test design that included both self-reported risk propensity and an actual risk-taking measure. We also used the full version of the Horne-Ostberg Morningness-Eveningness Questionnaire, which is a well-established criterion for measuring one aspect of circadian phenotype, diurnal preference. However, we did not include a measure of the sleep-wake chronotype as estimated from the Munich Chronotype Questionnaire. The study was limited in its age range as we sampled primarily young adults. Although our results apply to risk-taking in only a subset of the population, this subset includes individuals for which risk-taking is a serious public health concern. Our study includes only the health domain and we did not include other sub-domains of the DOSPERT. This may limit the interpretation of our results on risk propensity as they cannot be generalized for all domains of risk behavior. We did not include state anxiety or multiple risk propensity and risk-taking tasks.Our study provides strong support for the association between self-reported eveningness and higher risk propensity, as well as weaker support for higher risk-taking. We highlight a robust gender by chronotype effect where only females show a significant correlation between diurnal preference and risk-taking, with morning-type and intermediate-type females being more risk-averse than other individuals. Recent advances in chronobiology have highlighted major differences in how females and males respond physiologically to circadian factors ,55. ThesS1 TableComplete data for this paper is available in Excel spreadsheet format.(CSV)Click here for additional data file.