Expanding SPG18 clinical spectrum: autosomal dominant mutation causes complicated hereditary spastic paraplegia in a large family.

BACKGROUND: SPG18 is caused by mutations in the endoplasmic reticulum lipid raft associated 2 (ERLIN2) gene. Autosomal recessive (AR) mutations are usually associated with complicated hereditary spastic paraplegia (HSP), while autosomal dominant (AD) mutations use to cause pure SPG18. AIM: To define the variegate clinical spectrum of the SPG18 and to evaluate a dominant negative effect of erlin2 (encoded by ERLIN2) on oligomerization as causing differences between AR and AD phenotypes. METHODS: In a four-generation pedigree with an AD pattern, a spastic paraplegia multigene panel test was performed. Oligomerization of erlin2 was analyzed with velocity gradient assay in fibroblasts of the proband and healthy subjects. RESULTS: Despite the common p.V168M mutation identified in ERLIN2, a phenoconversion to amyotrophic lateral sclerosis (ALS) was observed in the second generation, pure HSP in the third generation, and a complicated form with psychomotor delay and epilepsy in the fourth generation. Erlin2 oligomerization was found to be normal. DISCUSSION: We report the first AD SPG18 family with a complicated phenotype, and we ruled out a dominant negative effect of V168M on erlin2 oligomerization. Therefore, our data do not support the hypothesis of a relationship between the mode of inheritance and the phenotype, but confirm the multifaceted nature of SPG18 on both genetic and clinical point of view. Clinicians should be aware of the importance of conducting an in-depth clinical evaluation to unmask all the possible manifestations associated to an only apparently pure SPG18 phenotype. We confirm the genotype-phenotype correlation between V168M and ALS emphasizing the value of close follow-up.

Chronic exposure to l-BMAA cyanotoxin induces cytoplasmic TDP-43 accumulation and glial activation, reproducing an amyotrophic lateral sclerosis-like phenotype in mice.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressive and often fatal neurodegenerative disease characterized by the loss of Motor Neurons (MNs) in spinal cord, motor cortex and brainstem. Despite significant efforts in the field, the exact pathogenetic mechanisms underlying both familial and sporadic forms of ALS have not been fully elucidated, and the therapeutic possibilities are still very limited. Here we investigate the molecular mechanisms of neurodegeneration induced by chronic exposure to the environmental cyanotoxin L-BMAA, which causes a form of ALS/Parkinson's disease (PD) in several populations consuming food and/or water containing high amounts of this compound. METHODS: In this effort, mice were chronically exposed to L-BMAA and analyzed at different time points to evaluate cellular and molecular alterations and behavioral deficits, performing MTT assay, immunoblot, immunofluorescence and immunohistochemistry analysis, and behavioral tests. RESULTS: We found that cyanotoxin L-BMAA determines apoptotic cell death and a marked astrogliosis in spinal cord and motor cortex, and induces neurotoxicity by favoring TDP-43 cytoplasmic accumulation. CONCLUSIONS: Overall, our results characterize a new versatile neurotoxic animal model of ALS that may be useful for the identification of new druggable targets to develop innovative therapeutic strategies for this disease.

Calcium-binding Cab45 regulates the polarized apical secretion of soluble proteins in epithelial cells.

Protein secretion is essential for epithelial tissue homoeostasis and therefore has to be tightly regulated. However, while the mechanisms regulating polarized protein sorting and trafficking have been widely studied in the past decade, those governing polarized secretion remain elusive. The calcium manganese pump SPCA1 and the calcium-binding protein Cab45 were recently shown to regulate the secretion of a subset of soluble cargoes in nonpolarized HeLa cells. Interestingly, we demonstrated that in polarized epithelial cells calcium levels in the trans-Golgi network (TGN), controlled by SPCA1, and Cab45 are critical for the apical sorting of glycosylphosphatidylinositol-anchored proteins (GPI-APs), a class of integral membrane proteins containing a soluble protein attached to the membrane by the GPI anchor, prompting us to investigate the mechanism regulating the polarized secretion of soluble cargoes. By reducing Cab45 expression level or overexpressing an inactive mutant of SPCA1, we found that Cab45 and calcium levels in the TGN drive the polarized apical secretion of a secretory form of placental alkaline phosphatase, exogenously expressed, and the endogenous soluble protein clusterin/Gp80 in Madin-Darby canine kidney (MDCK) cells. These data highlight the critical role of a calcium-dependent Cab45 mechanism regulating apical exocytosis in polarized MDCK cells.

Genetics and Molecular Basis of Congenital Heart Defects in Down Syndrome: Role of Extracellular Matrix Regulation.

Down syndrome (DS), a complex disorder that is caused by the trisomy of chromosome 21 (Hsa21), is a major cause of congenital heart defects (CHD). Interestingly, only about 50% of individuals with Hsa21 trisomy manifest CHD. Here we review the genetic basis of CHD in DS, focusing on genes that regulate extracellular matrix (ECM) organization. The overexpression of Hsa21 genes likely underlies the molecular mechanisms that contribute to CHD, even though the genes responsible for CHD could only be located in a critical region of Hsa21. A role in causing CHD has been attributed not only to protein-coding Hsa21 genes, but also to genes on other chromosomes, as well as miRNAs and lncRNAs. It is likely that the contribution of more than one gene is required, and that the overexpression of Hsa21 genes acts in combination with other genetic events, such as specific mutations or polymorphisms, amplifying their effect. Moreover, a key function in determining alterations in cardiac morphogenesis might be played by ECM. A large number of genes encoding ECM proteins are overexpressed in trisomic human fetal hearts, and many of them appear to be under the control of a Hsa21 gene, the RUNX1 transcription factor.

HIPK2 in cancer biology and therapy: Recent findings and future perspectives.

Homeodomain-interacting protein kinase 2 (HIPK2) is a serine-threonine kinase that phosphorylates and regulates a plethora of transcriptional regulators and chromatin modifiers. The heterogeneity of its interactome allows HIPK2 to modulate several cellular processes and signaling pathways, ultimately regulating cell fate and proliferation. Because of its p53-dependent pro-apoptotic activity and its downregulation in many tumor types, HIPK2 is traditionally considered a bone fide tumor suppressor gene. However, recent findings revealed that the role of HIPK2 in the pathogenesis of cancer is much more complex, ranging from tumor suppressive to oncogenic, strongly depending on the cellular context. Here, we review the very recent data emerged in the last years about the involvement of HIPK2 in cancer biology and therapy, highlighting the various alterations of this kinase (downregulation, upregulation, mutations and/or delocalization) in dependence on the cancer types. In addition, we discuss the recent advancement in the understanding the tumor suppressive and oncogenic functions of HIPK2, its role in establishing the response to cancer therapies, and its regulation by cancer-associated microRNAs. All these data strengthen the idea that HIPK2 is a key player in many types of cancer; therefore, it could represent an important prognostic marker, a factor to predict therapy response, and even a therapeutic target itself.

Truncated Analogues of a G-Quadruplex-Forming Aptamer Targeting Mutant Huntingtin: Shorter Is Better!

Two analogues of the MS3 aptamer, which was previously shown to have an exquisite capability to selectively bind and modulate the activity of mutant huntingtin (mHTT), have been here designed and evaluated in their physicochemical and biological properties. Featured by a distinctive propensity to form complex G-quadruplex structures, including large multimeric aggregates, the original 36-mer MS3 has been truncated to give a 33-mer (here named MS3-33) and a 17-mer (here named MS3-17). A combined use of different techniques (UV, CD, DSC, gel electrophoresis) allowed a detailed physicochemical characterization of these novel G-quadruplex-forming aptamers, tested in vitro on SH-SY5Y cells and in vivo on a Drosophila Huntington's disease model, in which these shorter MS3-derived oligonucleotides proved to have improved bioactivity in comparison with the parent aptamer.

Cocoa Extract Provides Protection against 6-OHDA Toxicity in SH-SY5Y Dopaminergic Neurons by Targeting PERK.

Parkinson's disease (PD) represents one of the most common neurodegenerative disorders, characterized by a dopamine (DA) deficiency in striatal synapses and misfolded toxic α-synuclein aggregates with concomitant cytotoxicity. In this regard, the misfolded proteins accumulation in neurodegenerative disorders induces a remarkable perturbations of endoplasmic reticulum (ER) homeostasis leading to persistent ER stress, which in turn, effects protein synthesis, modification, and folding quality control. A large body of evidence suggests that natural products target the ER stress signaling pathway, exerting a potential action in cancers, diabetes, cardiovascular and neurodegenerative diseases. This study aims to assess the neuroprotective effect of cocoa extract and its purified fractions against a cellular model of Parkinson's disease represented by 6-hydroxydopamine (6-OHDA)-induced SH-SY5Y human neuroblastoma. Our findings demonstrate, for the first time, the ability of cocoa to specifically targets PERK sensor, with significant antioxidant and antiapoptotic activities as both crude and fractioning extracts. In addition, cocoa also showed antiapoptotic properties in 3D cell model and a notable ability to inhibit the accumulation of α-synuclein in 6-OHDA-induced cells. Overall, these results indicate that cocoa exerts neuroprotective effects suggesting a novel possible strategy to prevent or, at least, mitigate neurodegenerative disorders, such as PD.

Genotype-Phenotype Correlations in Neurofibromatosis Type 1: Identification of Novel and Recurrent NF1 Gene Variants and Correlations with Neurocognitive Phenotype.

Neurofibromatosis type 1 (NF1) is one of the most common genetic tumor predisposition syndrome, caused by mutations in the NF1. To date, few genotype-phenotype correlations have been discerned in NF1, due to a highly variable clinical presentation. We aimed to study the molecular spectrum of NF1 and genotype-phenotype correlations in a monocentric study cohort of 85 NF1 patients (20 relatives, 65 sporadic cases). Clinical data were collected at the time of the mutation analysis and reviewed for accuracy in this investigation. An internal phenotypic categorization was applied. The 94% of the patients enrolled showed a severe phenotype with at least one systemic complication and a wide range of associated malignancies. Spine deformities were the most common complications in this cohort. We also reported 66 different NF1 mutations, of which 7 are novel mutations. Correlation analysis identified a slight significant inverse correlation between age at diagnosis and delayed acquisition of psychomotor skills with residual multi-domain cognitive impairment. Odds ratio with 95% confidence interval showed a higher prevalence of learning disabilities in patients carrying frameshift mutations. Overall, our results aim to offer an interesting contribution to studies on the genotype-phenotype of NF1 and in genetic management and counselling.

Down Syndrome Fetal Fibroblasts Display Alterations of Endosomal Trafficking Possibly due to SYNJ1 Overexpression.

Endosomal trafficking is essential for cellular homeostasis. At the crossroads of distinct intracellular pathways, the endolysosomal system is crucial to maintain critical functions and adapt to the environment. Alterations of endosomal compartments were observed in cells from adult individuals with Down syndrome (DS), suggesting that the dysfunction of the endosomal pathway may contribute to the pathogenesis of DS. However, the nature and the degree of impairment, as well as the timing of onset, remain elusive. Here, by applying imaging and biochemical approaches, we demonstrate that the structure and dynamics of early endosomes are altered in DS cells. Furthermore, we found that recycling trafficking is markedly compromised in these cells. Remarkably, our results in 18-20 week-old human fetal fibroblasts indicate that alterations in the endolysosomal pathway are already present early in development. In addition, we show that overexpression of the polyphosphoinositide phosphatase synaptojanin 1 (Synj1) recapitulates the alterations observed in DS cells, suggesting a role for this lipid phosphatase in the pathogenesis of DS, likely already early in disease development. Overall, these data strengthen the link between the endolysosomal pathway and DS, highlighting a dangerous liaison among Synj1, endosomal trafficking and DS.

Fighting the Huntington's Disease with a G-Quadruplex-Forming Aptamer Specifically Binding to Mutant Huntingtin Protein: Biophysical Characterization, In Vitro and In Vivo Studies.

A set of guanine-rich aptamers able to preferentially recognize full-length huntingtin with an expanded polyglutamine tract has been recently identified, showing high efficacy in modulating the functions of the mutated protein in a variety of cell experiments. We here report a detailed biophysical characterization of the best aptamer in the series, named MS3, proved to adopt a stable, parallel G-quadruplex structure and show high nuclease resistance in serum. Confocal microscopy experiments on HeLa and SH-SY5Y cells, as models of non-neuronal and neuronal cells, respectively, showed a rapid, dose-dependent uptake of fluorescein-labelled MS3, demonstrating its effective internalization, even in the absence of transfecting agents, with no general cytotoxicity. Then, using a well-established Drosophila melanogaster model for Huntington's disease, which expresses the mutated form of human huntingtin, a significant improvement in the motor neuronal function in flies fed with MS3 was observed, proving the in vivo efficacy of this aptamer.

Overexpression of the Hsa21 Transcription Factor RUNX1 Modulates the Extracellular Matrix in Trisomy 21 Cells.

Down syndrome is a neurodevelopmental disorder frequently characterized by other developmental defects, such as congenital heart disease. Analysis of gene expression profiles of hearts from trisomic fetuses have shown upregulation of extracellular matrix (ECM) genes. The aim of this work was to identify genes on chromosome 21 potentially responsible for the upregulation of ECM genes and to pinpoint any functional consequences of this upregulation. By gene set enrichment analysis of public data sets, we identified the transcription factor RUNX1, which maps to chromosome 21, as a possible candidate for regulation of ECM genes. We assessed that approximately 80% of ECM genes overexpressed in trisomic hearts have consensus sequences for RUNX1 in their promoters. We found that in human fetal fibroblasts with chromosome 21 trisomy there is increased expression of both RUNX1 and several ECM genes, whether located on chromosome 21 or not. SiRNA silencing of RUNX1 reduced the expression of 11 of the 14 ECM genes analyzed. In addition, collagen IV, an ECM protein secreted in high concentrations in the culture media of trisomic fibroblasts, was modulated by RUNX1 silencing. Attenuated expression of RUNX1 increased the migratory capacity of trisomic fibroblasts, which are characterized by a reduced migratory capacity compared to euploid controls.

Deregulation of microtubule organization and RNA metabolism in Arx models for lissencephaly and developmental epileptic encephalopathy.

X-linked lissencephaly with abnormal genitalia (XLAG) and developmental epileptic encephalopathy-1 (DEE1) are caused by mutations in the Aristaless-related homeobox (ARX) gene, which encodes a transcription factor responsible for brain development. It has been unknown whether the phenotypically diverse XLAG and DEE1 phenotypes may converge on shared pathways. To address this question, a label-free quantitative proteomic approach was applied to the neonatal brain of Arx knockout (ArxKO/Y) and knock-in polyalanine (Arx(GCG)7/Y) mice that are respectively models for XLAG and DEE1. Gene ontology and protein-protein interaction analysis revealed that cytoskeleton, protein synthesis and splicing control are deregulated in an allelic-dependent manner. Decreased α-tubulin content was observed both in Arx mice and Arx/alr-1(KO) Caenorhabditis elegans ,and a disorganized neurite network in murine primary neurons was consistent with an allelic-dependent secondary tubulinopathy. As distinct features of Arx(GCG)7/Y mice, we detected eIF4A2 overexpression and translational suppression in cortex and primary neurons. Allelic-dependent differences were also established in alternative splicing (AS) regulated by PUF60 and SAM68. Abnormal AS repertoires in Neurexin-1, a gene encoding multiple pre-synaptic organizers implicated in synaptic remodelling, were detected in Arx/alr-1(KO) animals and in Arx(GCG)7/Y epileptogenic brain areas and depolarized cortical neurons. Consistent with a conserved role of ARX in modulating AS, we propose that the allelic-dependent secondary synaptopathy results from an aberrant Neurexin-1 repertoire. Overall, our data reveal alterations mirroring the overlapping and variant effects caused by null and polyalanine expanded mutations in ARX. The identification of these effects can aid in the design of pathway-guided therapy for ARX endophenotypes and NDDs with overlapping comorbidities.

Phenotypic Effects of Homeodomain-Interacting Protein Kinase 2 Deletion in Mice.

Homeodomain-interacting protein kinase 2 (HIPK2) is a serine-threonine kinase that phosphorylates various transcriptional and chromatin regulators, thus modulating numerous important cellular processes, such as proliferation, apoptosis, DNA damage response, and oxidative stress. The role of HIPK2 in the pathogenesis of cancer and fibrosis is well established, and evidence of its involvement in the homeostasis of multiple organs has been recently emerging. We have previously demonstrated that Hipk2-null (Hipk2-KO) mice present cerebellar alterations associated with psychomotor abnormalities and that the double ablation of HIPK2 and its interactor HMGA1 causes perinatal death due to respiratory failure. To identify other alterations caused by the loss of HIPK2, we performed a systematic morphological analysis of Hipk2-KO mice. Post-mortem examinations and histological analysis revealed that Hipk2 ablation causes neuronal loss, neuronal morphological alterations, and satellitosis throughout the whole central nervous system (CNS); a myopathic phenotype characterized by variable fiber size, mitochondrial proliferation, sarcoplasmic inclusions, morphological alterations at neuromuscular junctions; and a cardiac phenotype characterized by fibrosis and cardiomyocyte hypertrophy. These data demonstrate the importance of HIPK2 in the physiology of skeletal and cardiac muscles and of different parts of the CNS, thus suggesting its potential relevance for different new aspects of human pathology.

Calcium levels in the Golgi complex regulate clustering and apical sorting of GPI-APs in polarized epithelial cells.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are lipid-associated luminal secretory cargoes selectively sorted to the apical surface of the epithelia where they reside and play diverse vital functions. Cholesterol-dependent clustering of GPI-APs in the Golgi is the key step driving their apical sorting and their further plasma membrane organization and activity; however, the specific machinery involved in this Golgi event is still poorly understood. In this study, we show that the formation of GPI-AP homoclusters (made of single GPI-AP species) in the Golgi relies directly on the levels of calcium within cisternae. We further demonstrate that the TGN calcium/manganese pump, SPCA1, which regulates the calcium concentration within the Golgi, and Cab45, a calcium-binding luminal Golgi resident protein, are essential for the formation of GPI-AP homoclusters in the Golgi and for their subsequent apical sorting. Down-regulation of SPCA1 or Cab45 in polarized epithelial cells impairs the oligomerization of GPI-APs in the Golgi complex and leads to their missorting to the basolateral surface. Overall, our data reveal an unexpected role for calcium in the mechanism of GPI-AP apical sorting in polarized epithelial cells and identify the molecular machinery involved in the clustering of GPI-APs in the Golgi.

Human Trisomic iPSCs from Down Syndrome Fibroblasts Manifest Mitochondrial Alterations Early during Neuronal Differentiation.

BACKGROUND: The presence of mitochondrial alterations in Down syndrome suggests that it might affect neuronal differentiation. We established a model of trisomic iPSCs, differentiating into neural precursor cells (NPCs) to monitor the occurrence of differentiation defects and mitochondrial dysfunction. METHODS: Isogenic trisomic and euploid iPSCs were differentiated into NPCs in monolayer cultures using the dual-SMAD inhibition protocol. Expression of pluripotency and neural differentiation genes was assessed by qRT-PCR and immunofluorescence. Meta-analysis of expression data was performed on iPSCs. Mitochondrial Ca2+, reactive oxygen species (ROS) and ATP production were investigated using fluorescent probes. Oxygen consumption rate (OCR) was determined by Seahorse Analyzer. RESULTS: NPCs at day 7 of induction uniformly expressed the differentiation markers PAX6, SOX2 and NESTIN but not the stemness marker OCT4. At day 21, trisomic NPCs expressed higher levels of typical glial differentiation genes. Expression profiles indicated that mitochondrial genes were dysregulated in trisomic iPSCs. Trisomic NPCs showed altered mitochondrial Ca2+, reduced OCR and ATP synthesis, and elevated ROS production. CONCLUSIONS: Human trisomic iPSCs can be rapidly and efficiently differentiated into NPC monolayers. The trisomic NPCs obtained exhibit greater glial-like differentiation potential than their euploid counterparts and manifest mitochondrial dysfunction as early as day 7 of neuronal differentiation.

PD-1 blockade delays tumor growth by inhibiting an intrinsic SHP2/Ras/MAPK signalling in thyroid cancer cells.

BACKGROUND: The programmed cell death-1 (PD-1) receptor and its ligands PD-L1 and PD-L2 are immune checkpoints that suppress anti-cancer immunity. Typically, cancer cells express the PD-Ls that bind PD-1 on immune cells, inhibiting their activity. Recently, PD-1 expression has also been found in cancer cells. Here, we analysed expression and functions of PD-1 in thyroid cancer (TC). METHODS: PD-1 expression was evaluated by immunohistochemistry on human TC samples and by RT-PCR, western blot and FACS on TC cell lines. Proliferation and migration of TC cells in culture were assessed by BrdU incorporation and Boyden chamber assays. Biochemical studies were performed by western blot, immunoprecipitation, pull-down and phosphatase assays. TC cell tumorigenicity was assessed by xenotransplants in nude mice. RESULTS: Human TC specimens (47%), but not normal thyroids, displayed PD-1 expression in epithelial cells, which significantly correlated with tumour stage and lymph-node metastasis. PD-1 was also constitutively expressed on TC cell lines. PD-1 overexpression/stimulation promoted TC cell proliferation and migration. Accordingly, PD-1 genetic/pharmacologic inhibition caused the opposite effects. Mechanistically, PD-1 recruited the SHP2 phosphatase to the plasma membrane and potentiated its phosphatase activity. SHP2 enhanced Ras activation by dephosphorylating its inhibitory tyrosine 32, thus triggering the MAPK cascade. SHP2, BRAF and MEK were necessary for PD-1-mediated biologic functions. PD-1 inhibition decreased, while PD-1 enforced expression facilitated, TC cell xenograft growth in mice by affecting tumour cell proliferation. CONCLUSIONS: PD-1 circuit blockade in TC, besides restoring anti-cancer immunity, could also directly impair TC cell growth by inhibiting the SHP2/Ras/MAPK signalling pathway.

Corrigendum: Pioglitazone Improves Mitochondrial Organization and Bioenergetics in Down Syndrome Cells.

[This corrects the article DOI: 10.3389/fgene.2019.00606.].

ZSCAN4+ mouse embryonic stem cells have an oxidative and flexible metabolic profile.

Cultured mouse embryonic stem cells are a heterogeneous population with diverse differentiation potential. In particular, the subpopulation marked by Zscan4 expression has high stem cell potency and shares with 2 cell stage preimplantation embryos both genetic and epigenetic mechanisms that orchestrate zygotic genome activation. Although embryonic de novo genome activation is known to rely on metabolites, a more extensive metabolic characterization is missing. Here we analyze the Zscan4+ mouse stem cell metabolic phenotype associated with pluripotency maintenance and cell reprogramming. We show that Zscan4+ cells have an oxidative and adaptable metabolism, which, on one hand, fuels a high bioenergetic demand and, on the other hand, provides intermediate metabolites for epigenetic reprogramming. Our findings enhance our understanding of the metastable Zscan4+ stem cell state with potential applications in regenerative medicine.

Targeting Mitochondrial Network Architecture in Down Syndrome and Aging.

Mitochondria are organelles that mainly control energy conversion in the cell. In addition, they also participate in many relevant activities, such as the regulation of apoptosis and calcium levels, and other metabolic tasks, all closely linked to cell viability. Functionality of mitochondria appears to depend upon their network architecture that may dynamically pass from an interconnected structure with long tubular units, to a fragmented one with short separate fragments. A decline in mitochondrial quality, which presents itself as an altered structural organization and a function of mitochondria, has been observed in Down syndrome (DS), as well as in aging and in age-related pathologies. This review provides a basic overview of mitochondrial dynamics, from fission/fusion mechanisms to mitochondrial homeostasis. Molecular mechanisms determining the disruption of the mitochondrial phenotype in DS and aging are discussed. The impaired activity of the transcriptional co-activator PGC-1α/PPARGC1A and the hyperactivation of the mammalian target of rapamycin (mTOR) kinase are emerging as molecular underlying causes of these mitochondrial alterations. It is, therefore, likely that either stimulating the PGC-1α activity or inhibiting mTOR signaling could reverse mitochondrial dysfunction. Evidence is summarized suggesting that drugs targeting either these pathways or other factors affecting the mitochondrial network may represent therapeutic approaches to improve and/or prevent the effects of altered mitochondrial function. Overall, from all these studies it emerges that the implementation of such strategies may exert protective effects in DS and age-related diseases.

Cell-penetrating peptides: two faces of the same coin.

Cell-penetrating peptides (CPPs) are short peptides able to cross the cellular membranes without any interaction with specific receptors. Thanks to their ability to transport various cargo inside the cells are emerged as powerful therapeutic agents alternative to small molecules. In recent years, numerous preclinical studies provided promising results for the treatment of various human diseases. Several CPP-conjugated compounds are under clinical trials.