Are Melanocortin Receptors Present in Extant Protochordates?

Functional melanocortin receptor (MCR) genes have been identified in the genomes of early chordates, e.g., the cyclostomata. Whether they appear in the most ancient chordates such as cephalochordate and urochordata, however, remains unclear due to missing genetic data. Herein, we studied five putative (from NCBI database), sequence-based predicted MCR-like receptors from urochordata and cephalochordate, including Styela clava, Ciona intestinalis, Branchiostoma floridae, and Branchiostoma belcheri. The BLAST and phylogenetic analyses suggested a relationship between these specific receptors and vertebrate MCRs. However, several essential residues for MCR functions in vertebrates were missing in these putative chordata MCRs. To test receptor functionality, several experimental studies were conducted. Binding assays and functional analyses showed no specific binding and no ligand-induced cAMP or ERK1/2 signaling (with either endogenous α-MSH or synthetic ligands for MC4R), despite successfully expressing four receptors in HEK 293T cells. These four receptors showed high basal cAMP signaling, likely mediated by ligand-independent Gs coupling. In summary, our results suggest that the five predicted MCR-like receptors are, indeed, class A G protein-coupled receptors (GPCRs), which in four cases show high constitutive activity in the Gs-cAMP signaling pathway but are not MCR-like receptors in terms of ligand recognition of known MCR ligands. These receptors might be ancient G protein-coupled receptors with so far unidentified ligands.

Ningalins, Pyrrole-Bearing Metabolites Isolated from Didemnum spp. Synthesis and MDR-Reversion Activity in Cancer Therapy.

Multi-Drug Resistance (MDR) is one of the most frequent problems observed in the course of cancer chemotherapy. Cells under treatment, tend to develop survival mechanisms to drug-action thus generating drug-resistance. One of the most important mechanism to get it is the over expression of P-gp glycoprotein, which acts as an efflux-pump releasing the drug outside of the cancer cell. A strategy for a succesfull treatment consists in the co-administration of one compound that acts against P-gp and another which acts against the cell during chemotherapy. Ningalins are pyrrole-containing naturally occurring compounds isolated mainly from the marine tunicate Didemnum spp and also they are some of the top reversing agents in MDR treatment acting on P-gp. Considering the relevance displayed for some of these isolated alkaloids or their core as a drug for co-administration in cancer therapy, all the total synthesis described to date for the members of ningalins family are reviewed herein.

Stem cell-mediated development, regeneration, chimerism, and aging in the colonial chordate Botryllus schlosseri.

Stem cells are units of biological organization, responsible for tissue and organ development and regeneration. I study stem cell biology, aging, and the evolution of immunity using the colonial chordate Botryllus schlosseri as a model system. This organism is uniquely suited for this study because it is closely related to vertebrates, undergoes weekly cycles of stem cell mediated regeneration, is long lived and has a recognition system and robust immune system. I have led the Botryllus genome project and developed a novel method to obtain a synthetic long read sequence, identified Botryllus stem cells and stem cell niches, isolated the gene that controls self/non self-recognition and characterized its immune system on the cellular and molecular levels. Recently, I led the Botryllus atlas project to characterize the two developmental pathways, embryogenesis (sexual) and blastogenesis (asexual), revealing the unique molecular landscapes for each developmental mode and investigated the molecular clock and neurodegeneration pathways in young and old colonies and investigated the molecular clock and neurodegeneration pathways in young and old colonies. These results and the resources we developed are used by my lab and others to further study stem cell and immune cell properties during development, regeneration, transplantation, and aging.

Increased collective migration correlates with germline stem cell competition in a basal chordate.

Cell competition is a process that compares the relative fitness of progenitor cells, resulting in winners, which contribute further to development, and losers, which are excluded, and is likely a universal quality control process that contributes to the fitness of an individual. Cell competition also has pathological consequences, and can create super-competitor cells responsible for tumor progression. We are studying cell competition during germline regeneration in the colonial ascidian, Botryllus schlosseri. Germline regeneration is due to the presence of germline stem cells (GSCs) which have a unique property: a competitive phenotype. When GSCs from one individual are transplanted into another, the donor and recipient cells compete for germline development. Often the donor GSCs win, and completely replace the gametes of the recipient- a process called germ cell parasitism (gcp). gcp is a heritable trait, and winner and loser genotypes can be found in nature and reared in the lab. However, the molecular and cellular mechanisms underlying gcp are unknown. Using an ex vivo migration assay, we show that GSCs isolated from winner genotypes migrate faster and in larger clusters than losers, and that cluster size correlates with expression of the Notch ligand, Jagged. Both cluster size and jagged expression can be manipulated simultaneously in a genotype dependent manner: treatment of loser GSCs with hepatocyte growth factor increases both jagged expression and cluster size, while inhibitors of the MAPK pathway decrease jagged expression and cluster size in winner GSCs. Live imaging in individuals transplanted with labeled winner and loser GSCs reveal that they migrate to the niche, some as small clusters, with the winners having a slight advantage in niche occupancy. Together, this suggests that the basis of GSC competition resides in a combination in homing ability and niche occupancy, and may be controlled by differential utilization of the Notch pathway.

Design of Balanced Cyclooxygenase Inhibitors Based on Natural Anti-inflammatory Ascidian Metabolites and Celecoxib.

The serious adverse effects caused by non-selective and selective cyclooxygenase-2 (COX-2) inhibitors remain significant concerns for current anti-inflammatory drugs. In this study, we present the design and synthesis of a novel series of celecoxib analogs incorporating a hydrazone linker, which were subjected to in silico analysis to compare their binding poses with those of clinically used nonsteroidal anti-inflammatory drugs (NSAIDs) against COX-1 and COX-2. The synthesized analogs were evaluated for their inhibitory activity against both COX enzymes, and compound 6 m, exhibiting potent balanced inhibition, was selected for subsequent in vitro anti-inflammatory assays. Treatment with 6 m effectively suppressed the NF-κB signaling pathway in lipopolysaccharide (LPS)-stimulated murine RAW264.7 macrophages, resulting in reduced expression of pro-inflammatory factors such as inducible nitric oxide synthase (iNOS), COX-2, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1ß, as well as decreased production of prostaglandin E2 (PGE2 ), nitric oxide (NO), and reactive oxygen species (ROS). However, 6 m has no effect on the MAPK signaling pathway. Therefore, due to its potent in vitro anti-inflammatory activity coupled with lack of cytotoxicity, 6 m represents a promising candidate for further development as a new lead compound targeting inflammation.

The effect of the sulfation patterns of dermatan and chondroitin sulfate from vertebrates and ascidians on their neuritogenic and neuroprotective properties.

Neurodegeneration is caused by the progressive loss of the structure and function of neurons, leading to cell death, and it is the main cause of many neurodegenerative diseases. Many molecules, such as glycosaminoglycans (GAGs), have been studied for their potential to prevent or treat these diseases. They are widespread in nature and perform an important role in neuritogenesis and neuroprotection. Here we investigated the neuritogenic and neuroprotective role of Phallusia nigra dermatan sulfate (PnD2,6S) and compared it with two distinct structures of chondroitin sulfate (C6S) and dermatan sulfate (D4S). For this study, a neuro 2A murine neuroblastoma cell line was used, and a chemical lesion was induced by the pesticide rotenone (ROT). We observed that PnD2,6S + ROT had a better neuritogenic effect than either C6S + ROT or D4S + ROT at a lower concentration (0.05 µg/mL). When evaluating the mitochondrial membrane potential, PnD2,6S showed a neuroprotective effect at a concentration of 0.4 µg/mL. These data indicate different mechanisms underlying this neuronal potential, in which the sulfation pattern is important for neuritogenic activity, while for neuroprotection all DS/CS structures had similar effects. This finding leads to a better understanding the chemical structures of PnD2,6S, C6S, and D4S and their therapeutic potential.

Ancestral glycoprotein hormone and its cognate receptor present in primitive chordate ascidian: Molecular identification and functional characterization.

The glycoprotein hormone (GPH) system is fundamentally significant in regulating the physiology of chordates, such as thyroid activity and gonadal function. However, the knowledge of the GPH system in the primitive chordate ascidian species is largely lacking. Here, we reported an ancestral GPH system in the ascidian (Styela clava), which consists of GPH α subunit (Sc-GPA2), GPH ß subunit (Sc-GPB5), and the cognate leucine-rich repeat-containing G protein-coupled receptor (Sc-GPHR). Comparative structure analysis revealed that distinct from vertebrate GPH ß subunits, Sc-GPB5 was less conserved, showing an atypical N-terminal sequence with a type II transmembrane domain instead of a typical signal peptide. By investigating the presence of recombinant Sc-GPA2 and Sc-GPB5 in cell lysates and culture media of HEK293T cells, we confirmed that these two subunits could be secreted out of the cells via distinct secretory pathways. The deglycosylation experiments demonstrated that N-linked glycosylation only occurred on the conserved cysteine residue (N78) of Sc-GPA2, whereas Sc-GPB5 was non-glycosylated. Although Sc-GPB5 exhibited distinct topology and biochemical properties in contrast to its chordate counterparts, it could still interact with Sc-GPA2 to form a heterodimer. The Sc-GPHR was then confirmed to be activated by tethered Sc-GPA2/GPB5 heterodimer on the Gs-cAMP pathway, suggesting that Sc-GPA2/GPB5 heterodimer-initiated Gs-cAMP signaling pathway is evolutionarily conserved in chordates. Furthermore, in situ hybridization and RT-PCR results revealed the co-expression patterns of Sc-GPA2 and Sc-GPB5 with Sc-GPHR transcripts, respectively in ascidian larvae and adults, highlighting the potential functions of Sc-GPA2/GPB5 heterodimer as an autocrine/paracrine neurohormone in regulating metamorphosis of larvae and physiological functions of adults. Our study systematically investigated the GPA2/GPB5-GPHR system in ascidian for the first time, which offers insights into understanding the function and evolution of the GPH system within the chordate lineage.

Localization of vasoactive intestinal peptide and toll-like receptor 2 immunoreactive cells in endostyle of urochordate Styela plicata (Lesueur, 1823).

The endostyle is the first component of the ascidian digestive tract, it is shaped like a through and is located in the pharynx's ventral wall. This organ is divided longitudinally into nine zones that are parallel to each other. Each zone's cells are physically and functionally distinct. Support elements are found in zones 1, 3, and 5, while mucoproteins secreting elements related to the filtering function are found in zones 2, 4, and 6. Zones 7, 8, and 9, which are located in the lateral dorsal section of the endostyle, include cells with high iodine and peroxidase concentrations. Immunohistochemical technique using the following antibodies, Toll-like receptor 2 (TLR-2) and vasoactive intestinal peptide (VIP), and lectin histochemistry (WGA-wheat-germagglutinin), were used in this investigation to define immune cells in the endostyle of Styela plicata (Lesueur, 1823). Our results demonstrate the presence of immune cells in the endostyle of S. plicata, highlighting that innate immune mechanisms are highly conserved in the phylogeny of the chordates. RESEARCH HIGHLIGHTS: Immune cells positive to TLR-2 and VIP in the endostyle of Styela plicata. Expression of WGA in several zones of endostyle. Use of comparative biology to improve the knowledge about immunology in ascidians.

Tunicates Illuminate the Enigmatic Evolution of Chordate Metallothioneins by Gene Gains and Losses, Independent Modular Expansions, and Functional Convergences.

To investigate novel patterns and processes of protein evolution, we have focused in the metallothioneins (MTs), a singular group of metal-binding, cysteine-rich proteins that, due to their high degree of sequence diversity, still represents a "black hole" in Evolutionary Biology. We have identified and analyzed more than 160 new MTs in nonvertebrate chordates (especially in 37 species of ascidians, 4 thaliaceans, and 3 appendicularians) showing that prototypic tunicate MTs are mono-modular proteins with a pervasive preference for cadmium ions, whereas vertebrate and cephalochordate MTs are bimodular proteins with diverse metal preferences. These structural and functional differences imply a complex evolutionary history of chordate MTs-including de novo emergence of genes and domains, processes of convergent evolution, events of gene gains and losses, and recurrent amplifications of functional domains-that would stand for an unprecedented case in the field of protein evolution.

Marine Natural Products from Tunicates and Their Associated Microbes.

Marine tunicates are identified as a potential source of marine natural products (MNPs), demonstrating a wide range of biological properties, like antimicrobial and anticancer activities. The symbiotic relationship between tunicates and specific microbial groups has revealed the acquisition of microbial compounds by tunicates for defensive purpose. For instance, yellow pigmented compounds, "tambjamines", produced by the tunicate, Sigillina signifera (Sluiter, 1909), primarily originated from their bacterial symbionts, which are involved in their chemical defense function, indicating the ecological role of symbiotic microbial association with tunicates. This review has garnered comprehensive literature on MNPs produced by tunicates and their symbiotic microbionts. Various sections covered in this review include tunicates' ecological functions, biological activities, such as antimicrobial, antitumor, and anticancer activities, metabolic origins, utilization of invasive tunicates, and research gaps. Apart from the literature content, 20 different chemical databases were explored to identify tunicates-derived MNPs. In addition, the management and exploitation of tunicate resources in the global oceans are detailed for their ecological and biotechnological implications.

Chemical Diversity and Biological Activity of Secondary Metabolites Isolated from Indonesian Marine Invertebrates.

Marine invertebrates have been reported to be an excellent resource of many novel bioactive compounds. Studies reported that Indonesia has remarkable yet underexplored marine natural products, with a high chemical diversity and a broad spectrum of biological activities. This review discusses recent updates on the exploration of marine natural products from Indonesian marine invertebrates (i.e., sponges, tunicates, and soft corals) throughout 2007-2020. This paper summarizes the structural diversity and biological function of the bioactive compounds isolated from Indonesian marine invertebrates as antimicrobial, antifungal, anticancer, and antiviral, while also presenting the opportunity for further investigation of novel compounds derived from Indonesian marine invertebrates.

Characterizing the cellular architecture of dynamically remodeling vascular tissue using 3-D image analysis and virtual reconstruction.

Epithelial tubules form critical structures in lung, kidney, and vascular tissues. However, the processes that control their morphogenesis and physiological expansion and contraction are not well understood. Here we examine the dynamic remodeling of epithelial tubes in vivo using a novel model system: the extracorporeal vasculature of Botryllus schlosseri, in which the disruption of the basement membrane triggers rapid, massive vascular retraction without loss of barrier function. We developed and implemented 3-D image analysis and virtual reconstruction tools to characterize the cellular morphology of the vascular wall in unmanipulated vessels and during retraction. In both control and regressed conditions, cells within the vascular wall were planar polarized, with an integrin- and curvature-dependent axial elongation of cells and a robust circumferential alignment of actin bundles. Surprisingly, we found no measurable differences in morphology between normal and retracting vessels under extracellular matrix (ECM) disruption. However, inhibition of integrin signaling through focal adhesion kinase inhibition caused disruption of cellular actin organization. Our results demonstrate that epithelial tubes can maintain tissue organization even during extreme remodeling events, but that the robust response to mechanical signals-such as the response to loss of vascular tension after ECM disruption-requires functional force sensing machinery via integrin signaling.

Origins and Bioactivities of Natural Compounds Derived from Marine Ascidians and Their Symbionts.

Marine ascidians are becoming important drug sources that provide abundant secondary metabolites with novel structures and high bioactivities. As one of the most chemically prolific marine animals, more than 1200 inspirational natural products, such as alkaloids, peptides, and polyketides, with intricate and novel chemical structures have been identified from ascidians. Some of them have been successfully developed as lead compounds or highly efficient drugs. Although numerous compounds that exist in ascidians have been structurally and functionally identified, their origins are not clear. Interestingly, growing evidence has shown that these natural products not only come from ascidians, but they also originate from symbiotic microbes. This review classifies the identified natural products from ascidians and the associated symbionts. Then, we discuss the diversity of ascidian symbiotic microbe communities, which synthesize diverse natural products that are beneficial for the hosts. Identification of the complex interactions between the symbiont and the host is a useful approach to discovering ways that direct the biosynthesis of novel bioactive compounds with pharmaceutical potentials.

Tunicate Heparan Sulfate Enriched in 2-Sulfated ß-Glucuronic Acid: Structure, Anticoagulant Activity, and Inhibitory Effect on the Binding of Human Colon Adenocarcinoma Cells to Immobilized P-Selectin.

Heparin or highly sulfated heparan sulfate (HS) has been described in different invertebrates. In ascidians (Chordata-Tunicata), these glycosaminoglycans occur in intracellular granules of oocyte accessory cells and circulating basophil-like cells, resembling mammalian mast cells and basophils, respectively. HS is also a component of the basement membrane of different ascidian organs. We have analyzed an HS isolated from the internal organs of the ascidian Phallusia nigra, using solution 1H/13C NMR spectroscopy, which allowed us to identify and quantify the monosaccharides found in this glycosaminoglycan. A variety of α-glucosamine units with distinct degrees of sulfation and N-acetylation were revealed. The hexuronic acid units occur both as α-iduronic acid and ß-glucuronic acid, with variable sulfation at the 2-position. A peculiar structural aspect of the tunicate HS is the high content of 2-sulfated ß-glucuronic acid, which accounts for one-third of the total hexuronic acid units. Another distinct aspect of this HS is the occurrence of high content of N-acetylated α-glucosamine units bearing a sulfate group at position 6. The unique ascidian HS is a potent inhibitor of the binding of human colon adenocarcinoma cells to immobilized P-selectin, being 11-fold more potent than mammalian heparin, but almost ineffective as an anticoagulant. Thus, the components of the HS structure required to inhibit coagulation and binding of tumor cells to P-selectin are distinct. Our results also suggest that the regulation of the pathway involved in the biosynthesis of glycosaminoglycans suffered variations during the evolution of chordates.

Anti-inflammatory effects of vanadium-binding protein from Halocynthia roretzi in LPS-stimulated RAW264.7 macrophages through NF-κB and MAPK pathways.

Vanadium-binding protein (VBP) was separated from the blood of the fresh sea urchin Halocynthia roretzi through (NH4)2SO4 precipitation, Diethylaminoethyl Sepharose fast-flow ion-exchange chromatography, and Sephacryl S-200 high-resolution size-exclusion chromatography. The protein size and purification yield of VBP were 27 kDa and 5.5%, respectively. VBP exerted anti-inflammatory effects in lipopolysaccharide-stimulated RAW264.7 macrophages by downregulating iNOS expression and inhibiting nitric oxide production. VBP also suppressed the expression of pro-inflammatory mediators such as COX-2, IL-1ß, IL-6, and TNF-α. The anti-inflammatory activity of VBP was further demonstrated in the NF-κB and MAPK inflammation pathways, in which VBP inhibited phosphorylation of signaling proteins such as p65, JNK, ERK1/2, and p38. Therefore, VBP from H. roretzi has anti-inflammatory effects and could potentially be used to treat inflammation.

Metallothioneins of the urochordate Oikopleura dioica have Cys-rich tandem repeats, large size and cadmium-binding preference.

The increasing levels of heavy metals derived from human activity are poisoning marine environments, threating zooplankton and ocean food webs. To protect themselves from the harmful effects of heavy metals, living beings have different physiological mechanisms, one of which is based on metallothioneins (MTs), a group of small cysteine-rich proteins that can bind heavy metals counteracting their toxicity. The MT system of urochordate appendicularians, an ecologically relevant component of the zooplankton, remained, however, unknown. In this work, we have characterized the MTs of the appendicularian species Oikopleura dioica, revealing that O. dioica has two MT genes, named OdMT1 and OdMT2, which encode for Cys-rich proteins, the former with 72 amino acids comparable with the small size MTs of other organisms, but the second with 399 amino acids representing the longest MT reported to date for any living being. Sequence analysis revealed that OdMT2 gene arose from a duplication of an ancestral OdMT1 gene followed by up to six tandem duplications of an ancestral repeat unit (RU) in the current OdMT2 gene. Interestingly, each RU contained, in turn, an internal repeat of a 7-Cys subunit (X3CX3CX2CX2CX3-6CX2CXCX), which is repeated up to 12 times in OdMT2. Finally, ICP-AES analyses of heterologously expressed OdMT proteins showed that both MTs were capable to form metal-complexes, with preference for cadmium ions. Collectively, our results provide the first characterization of the MT system in an appendicularian species as an initial step to understand the zooplankton response to metal toxicity and other environmental stress situations.

Immune-Enhancement and Anti-Inflammatory Activities of Fatty Acids Extracted from Halocynthia aurantium Tunic in RAW264.7 Cells.

Halocynthia aurantium, an edible ascidian species, has not been studied scientifically, even though tunicates and ascidians are well-known to contain several unique and biologically active materials. The current study investigated the fatty acid profiles of the H. aurantium tunic and its immune-regulatory effects on RAW264.7 macrophage cells. Results of the fatty acid profile analysis showed a difference in ratios, depending on the fatty acids being analysed, including those of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA). In particular, omega-3 fatty acids, such as eicosatrienoic acid n-3 (ETA n-3), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), were much higher than omega-6 fatty acids. Moreover, the H. aurantium tunic fatty acids, significantly and dose-dependently, increased the NO and prostaglandin E2 (PGE2) production in RAW264.7 cells, for immune-enhancement without cytotoxicity. In addition, these fatty acids regulated the transcription of immune-associated genes, including iNOS, IL-1ß, IL-6, COX-2, and TNF-α. These actions were activated and deactivated via Mitogen-activated protein kinase (MAPK)and NF-κB signaling, to regulate the immune responses. Conversely, the H. aurantium tunic fatty acids effectively suppressed the inflammatory cytokine expressions, including iNOS, IL-1ß, IL-6, COX-2, and TNF-α, in LPS-stimulated RAW264.7 cells. Productions of COX-2 and PGE2, which are key biomarkers for inflammation, were also significantly reduced. These results elucidated the immune-enhancement and anti-inflammatory mechanisms of the H. aurantium tunic fatty acids in macrophage cells. Moreover, the H. aurantium tunic might be a potential fatty acid source for immune-modulation.

Development of the house secreting epithelium, a major innovation of tunicate larvaceans, involves multiple homeodomain transcription factors.

The mechanisms driving innovations that distinguish large taxons are poorly known and essentially accessible via a candidate gene approach. A spectacular acquisition by tunicate larvaceans is the house, a complex extracellular filtration device. Its components are secreted by the oikoplastic epithelium which covers the animal trunk. Here we describe the development of this epithelium in larvae through the formation of specific cellular territories known to produce distinct sets of house proteins (Oikosins). It involves cell divisions and morphological differentiation but very limited cell migration. A diverse set of homeobox genes, most often duplicated in the genome, are transiently and site-specifically expressed in the trunk epithelium at early larval stages. Using RNA interference, we show that two prop duplicates are involved in the differentiation of a region on and around the dorsal midline, regulating morphology and the production of a specific oikosin. Our observations favor a scenario in which multiple homeobox genes and most likely other developmental transcription factors were recruited for this innovation. Their frequent duplications probably predated, but were not required for the emergence of the house.

Sameuramide A, a new cyclic depsipeptide isolated from an ascidian of the family Didemnidae.

Sameuramide A (1), a new cyclic depsipeptide encompassing one each of alanine, N-methyl alanine, N-methyl dehydroalanine, N,O-dimethyl threonine, phenyllactic acid, three ß-hydroxy leucines, and two propionates, was isolated from a didemnid ascidian collected at the northern part of Japan. The planar structure was established based on the interpretation of MS and NMR data. The absolute configuration of the subunits was determined by the advanced Marfey's method and the chiral LC-MS analysis. Compound 1 exhibited the activity of maintaining colony formation of murine embryonic stem (mES) cells without leukemia inhibitory factor (LIF). Down regulation of the gene expression of Krüppel-like transcription factor 4 (Klf4) indicated that 1 itself was not able to maintain the undifferentiated state of the mES cells. However, the expression levels of the marker genes (Nestin, T, Sox17) for three germ layers were upregulated in embryoid bodies (EBs) after treatment of 1 together with LIF, suggesting that 1 plays a supportive role for LIF in maintaining the multipotency of mES cells.

Ascidian Toxins with Potential for Drug Development.

Ascidians (tunicates) are invertebrate chordates, and prolific producers of a wide variety of biologically active secondary metabolites from cyclic peptides to aromatic alkaloids. Several of these compounds have properties which make them candidates for potential new drugs to treat diseases such as cancer. Many of these natural products are not produced by the ascidians themselves, rather by their associated symbionts. This review will focus mainly on the mechanism of action of important classes of cytotoxic molecules isolated from ascidians. These toxins affect DNA transcription, protein translation, drug efflux pumps, signaling pathways and the cytoskeleton. Two ascidian compounds have already found applications in the treatment of cancer and others are being investigated for their potential in cancer, neurodegenerative and other diseases.