Cortistatin as a Novel Multimodal Therapy for the Treatment of Parkinson's Disease.

Parkinson's disease (PD) is a complex disorder characterized by the impairment of the dopaminergic nigrostriatal system. PD has duplicated its global burden in the last few years, becoming the leading neurological disability worldwide. Therefore, there is an urgent need to develop innovative approaches that target multifactorial underlying causes to potentially prevent or limit disease progression. Accumulating evidence suggests that neuroinflammatory responses may play a pivotal role in the neurodegenerative processes that occur during the development of PD. Cortistatin is a neuropeptide that has shown potent anti-inflammatory and immunoregulatory effects in preclinical models of autoimmune and neuroinflammatory disorders. The goal of this study was to explore the therapeutic potential of cortistatin in a well-established preclinical mouse model of PD induced by acute exposure to the neurotoxin 1-methil-4-phenyl1-1,2,3,6-tetrahydropyridine (MPTP). We observed that treatment with cortistatin mitigated the MPTP-induced loss of dopaminergic neurons in the substantia nigra and their connections to the striatum. Consequently, cortistatin administration improved the locomotor activity of animals intoxicated with MPTP. In addition, cortistatin diminished the presence and activation of glial cells in the affected brain regions of MPTP-treated mice, reduced the production of immune mediators, and promoted the expression of neurotrophic factors in the striatum. In an in vitro model of PD, treatment with cortistatin also demonstrated a reduction in the cell death of dopaminergic neurons that were exposed to the neurotoxin. Taken together, these findings suggest that cortistatin could emerge as a promising new therapeutic agent that combines anti-inflammatory and neuroprotective properties to regulate the progression of PD at multiple levels.

Designing Antitrypanosomal and Antileishmanial BODIPY Derivatives: A Computational and In Vitro Assessment.

Leishmaniasis and Human African trypanosomiasis pose significant public health threats in resource-limited regions, accentuated by the drawbacks of the current antiprotozoal treatments and the lack of approved vaccines. Considering the demand for novel therapeutic drugs, a series of BODIPY derivatives with several functionalizations at the meso, 2 and/or 6 positions of the core were synthesized and characterized. The in vitro activity against Trypanosoma brucei and Leishmania major parasites was carried out alongside a human healthy cell line (MRC-5) to establish selectivity indices (SIs). Notably, the meso-substituted BODIPY, with 1-dimethylaminonaphthalene (1b) and anthracene moiety (1c), were the most active against L. major, displaying IC50 = 4.84 and 5.41 µM, with a 16 and 18-fold selectivity over MRC-5 cells, respectively. In contrast, the mono-formylated analogues 2b and 2c exhibited the highest toxicity (IC50 = 2.84 and 6.17 µM, respectively) and selectivity (SI = 24 and 11, respectively) against T. brucei. Further insights on the activity of these compounds were gathered from molecular docking studies. The results suggest that these BODIPYs act as competitive inhibitors targeting the NADPH/NADP+ linkage site of the pteridine reductase (PR) enzyme. Additionally, these findings unveil a range of quasi-degenerate binding complexes formed between the PRs and the investigated BODIPY derivatives. These results suggest a potential correlation between the anti-parasitic activity and the presence of multiple configurations that block the same site of the enzyme.

Synthesis and antimicrobial activity of aminoalkyl resveratrol derivatives inspired by cationic peptides.

Antimicrobial resistance is a global concern, far from being resolved. The need of new drugs against new targets is imminent. In this work, we present a family of aminoalkyl resveratrol derivatives with antibacterial activity inspired by the properties of cationic amphipathic antimicrobial peptides. Surprisingly, the newly designed molecules display modest activity against aerobically growing bacteria but show surprisingly good antimicrobial activity against anaerobic bacteria (Gram-negative and Gram-positive) suggesting specificity towards this bacterial group. Preliminary studies into the action mechanism suggest that activity takes place at the membrane level, while no cross-resistance with traditional antibiotics is observed. Actually, some good synergistic relations with existing antibiotics were found against Gram-negative pathogens. However, some cytotoxicity was observed, despite their low haemolytic activity. Our results show the importance of the balance between positively charged moieties and hydrophobicity to improve antimicrobial activity, setting the stage for the design of new drugs based on these molecules.

Polyaromatic Bis(indolyl)methane Derivatives with Antiproliferative and Antiparasitic Activity.

Bis(indolyl)methanes (BIMs) are a class of compounds that have been recognized as an important core in the design of drugs with important pharmacological properties, such as promising anticancer and antiparasitic activities. Here, we explored the biological activity of the BIM core functionalized with different (hetero)aromatic moieties. We synthesized substituted BIM derivatives with triphenylamine, N,N-dimethyl-1-naphthylamine and 8-hydroxylquinolyl groups, studied their photophysical properties and evaluated their in vitro antiproliferative and antiparasitic activities. The triphenylamine BIM derivative 2a displayed an IC50 of 3.21, 3.30 and 3.93 µM against Trypanosoma brucei, Leishmania major and HT-29 cancer cell line, respectively. The selectivity index demonstrated that compound 2a was up to eight-fold more active against the parasites and HT-29 than against the healthy cell line MRC-5. Fluorescence microscopy studies with MRC-5 cells and T. brucei parasites incubated with derivative 2a indicate that the compound seems to accumulate in the cell's mitochondria and in the parasite's nucleus. In conclusion, the BIM scaffold functionalized with the triphenylamine moiety proved to be the most promising antiparasitic and anticancer agent of this series.

Structure-activity relationship studies on divalent naphthalene diimide G quadruplex ligands with anticancer and antiparasitic activity.

Naphthalene diimide (NDI) is a central scaffold that has been commonly used in the design of G-quadruplex (G4) ligands. Previous work revealed notable anticancer activity of a disubstituted N-methylpiperazine propyl NDI G4 ligand. Here, we explored structure-activity relationship studies around ligand bis-N,N-2,7-(3-(4-methylpiperazin-1-yl)propyl)-1,4,5,8-naphthalenetetracarboxylic diimide, maintaining the central NDI core whilst modifying the spacer and the nature of the cationic groups. We prepared new disubstituted NDI derivatives of the original compound and examined their in vitro antiproliferative and antiparasitic activity. Several N-methylpiperazine propyl NDIs showed sub-micromolar activity against Trypanosoma brucei and Leishmania major parasites with up to 30 fold selectivity versus MRC-5 cells. The best compound was a dimorpholino NDI with an IC50 of 0.17 µM against T.brucei and 40 fold selectivity versus MRC-5 cells. However, no clear correlation between G4 binding of the new NDI derivatives and antiproliferative or antiparasitic activity was observed, indicating that other mechanisms of action may be responsible for the observed biological activity.

Chemically Tuning Resveratrol for the Effective Killing of Gram-Positive Pathogens.

In the era of antimicrobial resistance, the identification of new compounds with strong antimicrobial activity and the development of alternative therapies to fight drug-resistant bacteria are urgently needed. Here, we have used resveratrol, a safe and well-known plant-derived stilbene with poor antimicrobial properties, as a scaffold to design several new families of antimicrobials by adding different chemical entities at specific positions. We have characterized the mode of action of the most active compounds prepared and have examined their synergistic antibacterial activity in combination with traditional antibiotics. Some alkyl- and silyl-resveratrol derivatives show bactericidal activity against Gram-positive bacteria in the same low micromolar range of traditional antibiotics, with an original mechanism of action that combines membrane permeability activity with ionophore-related activities. No cross-resistance or antagonistic effect was observed with traditional antibiotics. Synergism was observed for some specific general-use antibiotics, such as aminoglycosides and cationic antimicrobial peptide antibiotics. No hemolytic activity was observed at the active concentrations or above, although some low toxicity against an MRC-5 cell line was noted.

Imide Condensation as a Strategy for the Synthesis of Core-Diversified G-Quadruplex Ligands with Anticancer and Antiparasitic Activity*.

A facile imide coupling strategy for the one-step preparation of G-quadruplex ligands with varied core chemistries is described. The G-quadruplex stabilization of a library of nine compounds was examined using FRET melting experiments, and CD, UV-Vis, fluorescence and NMR titrations, identifying several compounds that were capable of stabilizing G-quadruplex DNA with interesting selectivity profiles. The best G4 ligand was identified as compound 3, which was based on a perylene scaffold and exhibited 40-fold selectivity for a telomeric G-quadruplex over duplex DNA. Surprisingly, a tetra-substituted flexible core, compound 11, also exhibited selective stabilization of G4 DNA over duplex DNA. The anticancer and antiparasitic activity of the library was also examined, with the lead compound 3 exhibiting nanomolar inhibition of Trypanosoma brucei with 78-fold selectivity over MRC5 cells. The cellular localization of this compound was also studied via fluorescence microscopy. We found that uptake was time dependant, with localization outside the nucleus and kinetoplast that could be due to strong fluorescence quenching in the presence of small amounts of DNA.

Stiff-Stilbene Ligands Target G-Quadruplex DNA and Exhibit Selective Anticancer and Antiparasitic Activity.

G-quadruplex nucleic acid structures have long been studied as anticancer targets whilst their potential in antiparasitic therapy has only recently been recognized and barely explored. Herein, we report the synthesis, biophysical characterization, and in vitro screening of a series of stiff-stilbene G4 binding ligands featuring different electronics, side-chain chemistries, and molecular geometries. The ligands display selectivity for G4 DNA over duplex DNA and exhibit nanomolar toxicity against Trypasanoma brucei and HeLa cancer cells whilst remaining up to two orders of magnitude less toxic to non-tumoral mammalian cell line MRC-5. Our study demonstrates that stiff-stilbenes show exciting potential as the basis of selective anticancer and antiparasitic therapies. To achieve the most efficient G4 recognition the scaffold must possess the optimal electronics, substitution pattern and correct molecular configuration.

Symmetric and dissymmetric carbohydrate-phenyl ditriazole derivatives as DNA G-quadruplex ligands: Synthesis, biophysical studies and antiproliferative activity.

Here we present a novel G4-binding family of compounds based on a central core of phenyl ditriazole (PDTZ) modified with carbohydrates and phenyl pyrrolidinyl side-chains. Their synthesis was achieved using controlled click chemistry conditions to obtain both, symmetric and dissymmetric carb-PDTZ derivatives without any intermediate protecting steps through an optimized methodology. Binding of the new carb-PDTZ to a variety of G-quadruplex motifs was examined using different biophysical techniques. The symmetric carb-PDTZ derivatives were not able to stabilize G4, but the dissymmetric ones (containing one sugar and one phenyl pyrrolidinyl side-chain) did. Interestingly, the dissymmetric carb-PDTZ derivatives showed much higher G4 vs duplex DNA selectivity than the control compound PDTZ 1, which contains two phenyl pyrrodilinyl side-chains and no carbohydrates. Their potential antitumoral activity was also investigated by in vitro cytotoxicity measurements on different cancerous cell lines. All carb-PDTZ derivatives showed higher IC50 values than the control PDTZ 1, probably due to the lack of compound stability of some derivatives and to lower cellular uptake.

The Resveratrol Prodrug JC19 Delays Retinal Degeneration in rd10 Mice.

It has been reported that resveratrol (RES) has a therapeutic effect in different neurodegenerative and ocular diseases. However, RES is rapidly eliminated from the organism, and high doses need to be administered resulting in potential toxic side effects. We hypothesized that a RES prodrug such as 3,4'-diglucosyl resveratrol (JC19) would reduce RES metabolism to produce a neuroprotective effect. Here, we have examined the protective effect of JC19 in an experimental mouse model of autosomal recessive RP. Rd10 mice at postnatal day 13 (P13) were subretinally injected with vehicle and two different doses of JC19. Electroretinogram (ERG) and histological evaluation were performed 15 days after injections. The amplitude of a- and b-waves was quantified in ERG recordings, and the number of photoreceptor nuclei in the outer nuclear layer was counted. In addition, the mouse retinas were immunostained with anti-rhodopsin antibodies. JC19 treatment delayed the loss of rod photoreceptor in rd10 mice, maintaining the expression of rhodopsin and preserving their electrical responses to light stimuli. The exact mechanism by which RES delays retinal degeneration in rd10 mice remains to be elucidated, but Sirtuin 1 activation could be one of the key molecular pathways involved in its neuroprotective effect.

Enzymatic Synthesis of a Novel Pterostilbene α-Glucoside by the Combination of Cyclodextrin Glucanotransferase and Amyloglucosidase.

The synthesis of a novel α-glucosylated derivative of pterostilbene was performed by a transglycosylation reaction using starch as glucosyl donor, catalyzed by cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. The reaction was carried out in a buffer containing 20% (v/v) DMSO to enhance the solubility of pterostilbene. Due to the formation of several polyglucosylated products with CGTase, the yield of monoglucoside was increased by the treatment with a recombinant amyloglucosidase (STA1) from Saccharomyces cerevisiae (var. diastaticus). This enzyme was not able to hydrolyze the linkage between the glucose and pterostilbene. The monoglucoside was isolated and characterized by combining ESI-MS and 2D-NMR methods. Pterostilbene α-d-glucopyranoside is a novel compound. The α-glucosylation of pterostilbene enhanced its solubility in water to approximately 0.1 g/L. The α-glucosylation caused a slight loss of antioxidant activity towards ABTS˙⁺ radicals. Pterostilbene α-d-glucopyranoside was less toxic than pterostilbene for human SH-S5Y5 neurons, MRC5 fibroblasts and HT-29 colon cancer cells, and similar for RAW 264.7 macrophages.

The effect of l-thymidine, acyclic thymine and 8-bromoguanine on the stability of model G-quadruplex structures.

BACKGROUND: Guanine-rich oligonucleotides are capable of forming tetrahelical structures known as G-quadruplexes with interesting biological properties. We have investigated the effects of site-specific substitution in the loops and in the tetrads model G-quadruplexes using thymine glycol nucleic acid (GNA) units, l-thymidine and 8-Br-2'-deoxyguanosine. METHODS: Modified oligonucleotides were chemically synthesized and spectroscopic techniques were used to determine the relative stability of the modified G-quadruplex. The double 8-BrdG-modified quadruplexes were further characterized by Nuclear Magnetic Resonance. Binding to thrombin of selected quadruplex was analyzed by gel electrophoresis retention assay. RESULTS: The most interesting results were found with a 8-bromoG substitution that had the larger stabilization of the quadruplex. NMR studies indicate a tight relationship between the loops and the tetrads to accommodate 8-bromoG modifications within the TBA. CONCLUSIONS: The substitutions of loop positions with GNA T affect the TBA stability except for single modification in T7 position. Single l-thymidine substitutions produced destabilization of TBA. Larger changes on quadruplex stability are observed with the use of 8-bromoG finding a single substitution with the highest thermal stabilization found in thrombin binding aptamers modified at the guanine residues and having good affinity for thrombin. Double 8-BrdG modification in anti positions of different tetrads produce a conformational flip from syn to anti conformation of 8-Br-dG to favor loop-tetrad interaction and preserve the overall TBA stability. GENERAL SIGNIFICANCE: Modified guanine-rich oligonucleotides are valuable tools for the search for G-quadruplex structures with higher thermal stability and may provide compounds with interesting protein-nucleic acid binding properties. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

Cytotoxic, Antiangiogenic and Antitelomerase Activity of Glucosyl- and Acyl- Resveratrol Prodrugs and Resveratrol Sulfate Metabolites.

Resveratrol (RES) is a natural polyphenol with relevant and varied biological activity. However, its low bioavailability and rapid metabolism to its glucuronate and sulfate conjugates has opened a debate on the mechanisms underlying its bioactivity. RES prodrugs are being developed to overcome these problems. We have synthesized a series of RES prodrugs and RES sulfate metabolites (RES-S) and evaluated their biological activities. RES glucosylated prodrugs (RES-Glc) were more cytotoxic in HT-29 and MCF-7 cells than RES itself whereas RES-S showed similar or higher cytotoxicity than RES. VEGF production was decreased by RES-Glc, and RES-disulfate (RES-diS) diminished it even more than RES. Finally, RES-Glc and RES-diS inhibited hTERT gene expression to a higher extent than RES. In conclusion, resveratrol prodrugs are promising candidates as anticancer drugs. In addition, RES-S showed distinct biological activity, thus indicating they are not simply RES reservoirs.

Glucose-Nucleobase Pseudo Base Pairs: Biomolecular Interactions within DNA.

Noncovalent forces rule the interactions between biomolecules. Inspired by a biomolecular interaction found in aminoglycoside-RNA recognition, glucose-nucleobase pairs have been examined. Deoxyoligonucleotides with a 6-deoxyglucose insertion are able to hybridize with their complementary strand, thus exhibiting a preference for purine nucleobases. Although the resulting double helices are less stable than natural ones, they present only minor local distortions. 6-Deoxyglucose stays fully integrated in the double helix and its OH groups form two hydrogen bonds with the opposing guanine. This 6-deoxyglucose-guanine pair closely resembles a purine-pyrimidine geometry. Quantum chemical calculations indicate that glucose-purine pairs are as stable as a natural T-A pair.

Epigenetic therapy for Friedreich ataxia.

OBJECTIVE: To investigate whether a histone deacetylase inhibitor (HDACi) would be effective in an in vitro model for the neurodegenerative disease Friedreich ataxia (FRDA) and to evaluate safety and surrogate markers of efficacy in a phase I clinical trial in patients. METHODS: We used a human FRDA neuronal cell model, derived from patient induced pluripotent stem cells, to determine the efficacy of a 2-aminobenzamide HDACi (109) as a modulator of FXN gene expression and chromatin histone modifications. FRDA patients were dosed in 4 cohorts, ranging from 30mg/day to 240mg/day of the formulated drug product of HDACi 109, RG2833. Patients were monitored for adverse effects as well as for increases in FXN mRNA, frataxin protein, and chromatin modification in blood cells. RESULTS: In the neuronal cell model, HDACi 109/RG2833 increases FXN mRNA levels and frataxin protein, with concomitant changes in the epigenetic state of the gene. Chromatin signatures indicate that histone H3 lysine 9 is a key residue for gene silencing through methylation and reactivation through acetylation, mediated by the HDACi. Drug treatment in FRDA patients demonstrated increased FXN mRNA and H3 lysine 9 acetylation in peripheral blood mononuclear cells. No safety issues were encountered. INTERPRETATION: Drug exposure inducing epigenetic changes in neurons in vitro is comparable to the exposure required in patients to see epigenetic changes in circulating lymphoid cells and increases in gene expression. These findings provide a proof of concept for the development of an epigenetic therapy for this fatal neurological disease.

Effects of sugar functional groups, hydrophobicity, and fluorination on carbohydrate-DNA stacking interactions in water.

Carbohydrate-aromatic interactions are highly relevant for many biological processes. Nevertheless, experimental data in aqueous solution relating structure and energetics for sugar-arene stacking interactions are very scarce. Here, we evaluate how structural variations in a monosaccharide including carboxyl, N-acetyl, fluorine, and methyl groups affect stacking interactions with aromatic DNA bases. We find small differences on stacking interaction among the natural carbohydrates examined. The presence of fluorine atoms within the pyranose ring slightly increases the interaction with the C-G DNA base pair. Carbohydrate hydrophobicity is the most determinant factor. However, gradual increase in hydrophobicity of the carbohydrate does not translate directly into a steady growth in stacking interaction. The energetics correlates better with the amount of apolar surface buried upon sugar stacking on top of the aromatic DNA base pair.

Structure-Activity Study on Substituted, Core-Extended, and Dyad Naphthalene Diimide G-Quadruplex Ligands Leading to Potent Antitrypanosomal Agents.

Several G-quadruplex nucleic acid (G4s) ligands have been developed seeking target selectivity in the past decade. Naphthalene diimide (NDI)-based compounds are particularly promising due to their biological activity and red-fluorescence emission. Previously, we demonstrated the existence of G4s in the promoter region of parasite genomes, assessing the effectiveness of NDI-derivatives against them. Here, we explored the biological activity of a small library of G4-DNA ligands, exploiting the NDI pharmacophore, against both Trypanosoma brucei and Leishmania major parasites. Biophysical and biological assays were conducted. Among the various families analyzed, core-extended NDIs exhibited the most promising results concerning the selectivity and antiparasitic effects. NDI 16 emerged as the most potent, with an IC50 of 0.011 nM against T. brucei and remarkable selectivity vs MRC-5 cells (3454-fold). Fascinating, 16 is 480-fold more potent than the standard drug pentamidine (IC50 = 5.3 nM). Cellular uptake and parasite localization were verified by exploiting core-extended NDI red-fluorescent emission.

DNA G-quadruplexes in the genome of Trypanosoma cruzi as potential therapeutic targets for Chagas disease: Dithienylethene ligands as effective antiparasitic agents.

Chagas disease is caused by the parasite Trypanosoma cruzi and affects over 7 million people worldwide. The two actual treatments, Benznidazole (Bzn) and Nifurtimox, cause serious side effects due to their high toxicity leading to treatment abandonment by the patients. In this work, we propose DNA G-quadruplexes (G4) as potential therapeutic targets for this infectious disease. We have found 174 PQS per 100,000 nucleotides in the genome of T. cruzi and confirmed G4 formation of three frequent motifs. We synthesized a family of 14 quadruplex ligands based in the dithienylethene (DTE) scaffold and demonstrated their binding to these identified G4 sequences. Several DTE derivatives exhibited micromolar activity against epimastigotes of four different strains of T. cruzi, in the same concentration range as Bzn. Compounds L3 and L4 presented remarkable activity against trypomastigotes, the active form in blood, of T. cruzi SOL strain (IC50 = 1.5-3.3 µM, SI = 25-40.9), being around 40 times more active than Bzn and displaying much better selectivity indexes.

Piceid Octanoate Protects Retinal Cells against Oxidative Damage by Regulating the Sirtuin 1/Poly-ADP-Ribose Polymerase 1 Axis In Vitro and in rd10 Mice.

Retinitis pigmentosa is a common cause of inherited blindness in adults, which in many cases is associated with an increase in the formation of reactive oxygen species (ROS) that induces DNA damage, triggering Poly-ADP-Ribose Polymerase 1 (PARP1) activation and leading to parthanatos-mediated cell death. Previous studies have shown that resveratrol (RSV) is a promising molecule that can mitigate PARP1 overactivity, but its low bioavailability is a limitation for medical use. This study examined the impact of a synthesized new acylated RSV prodrug, piceid octanoate (PIC-OCT), in the 661W cell line against H2O2 oxidative stress and in rd10 mice. PIC-OCT possesses a better ADME profile than RSV. In response to H2O2, 661W cells pretreated with PIC-OCT preserved cell viability in more than 38% of cells by significantly promoting SIRT1 nuclear translocation, preserving NAD+/NADH ratio, and suppressing intracellular ROS formation. These effects result from expressing antioxidant genes, maintaining mitochondrial function, reducing PARP1 nuclear expression, and preventing AIF nuclear translocation. In rd10 mice, PIC-OCT inhibited PAR-polymer formation, increased SIRT1 expression, significantly reduced TUNEL-positive cells in the retinal outer nuclear layer, preserved ERGs, and enhanced light chamber activity (all p values < 0.05). Our findings corroborate that PIC-OCT protects photoreceptors by modulating the SIRT1/PARP1 axis in models of retinal degeneration.

Thiosugar naphthalene diimide conjugates: G-quadruplex ligands with antiparasitic and anticancer activity.

Glycosyl conjugation to drugs is a strategy being used to take advantage of glucose transporters (GLUT) overexpression in cancer cells in comparison with non-cancerous cells. Its extension to the conjugation of drugs to thiosugars tries to exploit their higher biostability when compared to O-glycosides. Here, we have synthesized a series of thiosugar naphthalene diimide conjugates as G-quadruplex ligands and have explored modifications of the amino sidechain comparing dimethyl amino and morpholino groups. Then, we studied their antiproliferative activity in colon cancer cells, and their antiparasitic activity in T. brucei and L. major parasites, together with their ability to bind quadruplexes and their cellular uptake and location. We observed higher toxicity for the sugar-NDI-NMe2 derivatives than for the sugar-NDI-morph compounds, both in mammalian cells and in parasites. Our experiments indicate that a less efficient binding to quadruplexes and a worse cellular uptake of the carb-NDI-morph derivatives could be the reasons for these differences. We found small variations in cytotoxicity between O-carb-NDIs and S-carb-NDIs, except against non-cancerous human fibroblasts MRC-5, where thiosugar-NDIs tend to be less toxic. This leads to a notable selectivity for ß-thiomaltosyl-NDI-NMe212 (9.8 fold), with an IC50 of 0.3 µM against HT-29 cells. Finally, the antiparasitic activity observed for the carb-NDI-NMe2 derivatives against T. brucei was in the nanomolar range with a good selectivity index in the range of 30- to 69- fold.