Ligand co-crystallization of aminoacyl-tRNA synthetases from infectious disease organisms.

Aminoacyl-tRNA synthetases (aaRSs) charge tRNAs with their cognate amino acid, an essential precursor step to loading of charged tRNAs onto the ribosome and addition of the amino acid to the growing polypeptide chain during protein synthesis. Because of this important biological function, aminoacyl-tRNA synthetases have been the focus of anti-infective drug development efforts and two aaRS inhibitors have been approved as drugs. Several researchers in the scientific community requested aminoacyl-tRNA synthetases to be targeted in the Seattle Structural Genomics Center for Infectious Disease (SSGCID) structure determination pipeline. Here we investigate thirty-one aminoacyl-tRNA synthetases from infectious disease organisms by co-crystallization in the presence of their cognate amino acid, ATP, and/or inhibitors. Crystal structures were determined for a CysRS from Borrelia burgdorferi bound to AMP, GluRS from Borrelia burgdorferi and Burkholderia thailandensis bound to glutamic acid, a TrpRS from the eukaryotic pathogen Encephalitozoon cuniculi bound to tryptophan, a HisRS from Burkholderia thailandensis bound to histidine, and a LysRS from Burkholderia thailandensis bound to lysine. Thus, the presence of ligands may promote aaRS crystallization and structure determination. Comparison with homologous structures shows conformational flexibility that appears to be a recurring theme with this enzyme class.

Dual 5' Cap Labeling Based on Regioselective RNA Methyltransferases and Bioorthogonal Reactions.

The ability to detect and localize defined RNA strands inside living cells requires probes with high specificity, sensitivity, and signal-to-background ratio. To track low-abundant biomolecules, such as strands of regular mRNA, and distinguish fluorescence signal from the background after bioorthogonal reactions in cells, it is imperative to employ turn-on concepts. Here, we have presented a straightforward enzymatic approach to allow site-specific modification of two different positions on the 5' cap of eukaryotic mRNA with either identical or different small functional groups. The approach relies on two methyltransferases and analogues of their natural co-substrate, and it can be extended to a three-enzyme cascade reaction for their in situ production. Subsequent labeling by using bioorthogonal click reactions provided access to double labeling with identical fluorophores or dual labeling with two different reporter groups, as exemplified by a Cy5 dye, a FRET pair, and a fluorophore/biotin combination. Our dual-labeling strategy addresses the need for increased sensitivity and should improve the signal-to-background ratio after bioorthogonal reactions in cells.

Modifying the 5'-Cap for Click Reactions of Eukaryotic mRNA and To Tune Translation Efficiency in Living Cells.

The 5'-cap is a hallmark of eukaryotic mRNAs and plays fundamental roles in RNA metabolism, ranging from quality control to export and translation. Modifying the 5'-cap may thus enable modulation of the underlying processes and investigation or tuning of several biological functions. A straightforward approach is presented for the efficient production of a range of N7-modified caps based on the highly promiscuous methyltransferase Ecm1. We show that these, as well as N(2) -modified 5'-caps, can be used to tune translation of the respective mRNAs both in vitro and in cells. Appropriate modifications allow subsequent bioorthogonal chemistry, as demonstrated by intracellular live-cell labeling of a target mRNA. The efficient and versatile N7 manipulation of the mRNA cap makes mRNAs amenable to both modulation of their biological function and intracellular labeling, and represents a valuable addition to the chemical biology toolbox.

ATP-dependent conformational dynamics underlie the functional asymmetry of the replicative helicase from a minimalist eukaryote.

The heterohexameric minichromosome maintenance (MCM2-7) complex is an ATPase that serves as the central replicative helicase in eukaryotes. During initiation, the ring-shaped MCM2-7 particle is thought to open to facilitate loading onto DNA. The conformational state accessed during ring opening, the interplay between ATP binding and MCM2-7 architecture, and the use of these events in the regulation of DNA unwinding are poorly understood. To address these issues in isolation from the regulatory complexity of existing eukaryotic model systems, we investigated the structure/function relationships of a naturally minimized MCM2-7 complex from the microsporidian parasite Encephalitozoon cuniculi. Electron microscopy and small-angle X-ray scattering studies show that, in the absence of ATP, MCM2-7 spontaneously adopts a left-handed, open-ring structure. Nucleotide binding does not promote ring closure but does cause the particle to constrict in a two-step process that correlates with the filling of high- and low-affinity ATPase sites. Our findings support the idea that an open ring forms the default conformational state of the isolated MCM2-7 complex, and they provide a structural framework for understanding the multiphasic ATPase kinetics observed in different MCM2-7 systems.

Structure of fructose bisphosphate aldolase from Encephalitozoon cuniculi.

Fructose bisphosphate aldolose (FBPA) enzymes have been found in a broad range of eukaryotic and prokaryotic organisms. FBPA catalyses the cleavage of fructose 1,6-bisphosphate into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. The SSGCID has reported several FBPA structures from pathogenic sources. Bioinformatic analysis of the genome of the eukaryotic microsporidian parasite Encephalitozoon cuniculi revealed an FBPA homolog. The structures of this enzyme in the presence of the native substrate FBP and also with the partial substrate analog phosphate are reported. The purified enzyme crystallized in 90 mM Bis-Tris propane pH 6.5, 18% PEG 3350, 18 mM NaKHPO(4), 10 mM urea for the phosphate-bound form and 100 mM Bis-Tris propane pH 6.5, 20% PEG 3350, 20 mM fructose 1,6-bisphosphate for the FBP-bound form. In both cases protein was present at 25 mg ml(-1) and the sitting-drop vapour-diffusion method was used. For the FBP-bound form, a data set to 2.37 Å resolution was collected from a single crystal at 100 K. The crystal belonged to the orthorhombic space group C222(1), with unit-cell parameters a=121.46, b=135.82, c=61.54 Å. The structure was refined to a final free R factor of 20.8%. For the phosphate-bound form, a data set was collected to 2.00 Å resolution. The space group was also C222(1) and the unit-cell parameters were a=121.96, b=137.61, c=62.23 Å. The structure shares the typical barrel tertiary structure reported for previous FBPA structures and exhibits the same Schiff base in the active site. The quaternary structure is dimeric. This work provides a direct experimental result for the substrate-binding conformation of the product state of E. cuniculi FBPA.

Molecular structure of the N-terminal domain of the APC/C subunit Cdc27 reveals a homo-dimeric tetratricopeptide repeat architecture.

The anaphase promoting complex/cyclosome (APC/C) is a large multi-subunit E3 ubiquitin ligase that targets specific cell cycle regulatory proteins for ubiquitin-dependent degradation, thereby controlling cell cycle events such as the metaphase to anaphase transition and the exit from mitosis. Biochemical and genetic studies are consistent with the notion that subunits of APC/C are organised into two distinct sub-complexes; a catalytic sub-complex including the cullin domain and RING finger subunits Apc2 and Apc11, respectively, and a tetratricopeptide repeat (TPR) sub-complex composed of the TPR subunits Cdc16, Cdc23 and Cdc27 (Apc3). Here, we describe the crystal structure of the N-terminal domain of Encephalitozoon cuniculi Cdc27 (Cdc27(Nterm)), revealing a homo-dimeric structure, composed predominantly of successive TPR motifs. Mutation of the Cdc27(Nterm) dimer interface destabilises the protein, disrupts dimerisation in solution, and abolishes the capacity of E. cuniculi Cdc27 to complement Saccharomyces cerevisiae Cdc27 in vivo. These results establish the existence of functional APC/C genes in E. cuniculi, the evolutionarily conserved dimeric properties of Cdc27, and provide a framework for understanding the architecture of full-length Cdc27.

Structure of a microsporidian methionine aminopeptidase type 2 complexed with fumagillin and TNP-470.

Microsporidia are protists that have been reported to cause infections in both vertebrates and invertebrates. They have emerged as human pathogens particularly in patients that are immunosuppressed and cases of gastrointestinal infection, encephalitis, keratitis, sinusitis, myositis and disseminated infection are well described in the literature. While benzimidazoles are active against many species of microsporidia, these drugs do not have significant activity against Enterocytozoon bieneusi. Fumagillin and its analogues have been demonstrated to have activity invitro and in animal models of microsporidiosis and human infections due to E. bieneusi. Fumagillin and its analogues inhibit methionine aminopeptidase type 2. Encephalitozoon cuniculi MetAP2 (EcMetAP2) was cloned and expressed as an active enzyme using a baculovirus system. The crystal structure of EcMetAP2 was determined with and without the bound inhibitors fumagillin and TNP-470. This structure classifies EcMetAP2 as a member of the MetAP2c family. The EcMetAP2 structure was used to generate a homology model of the E. bieneusi MetAP2. Comparison of microsporidian MetAP2 structures with human MetAP2 provides insights into the design of inhibitors that might exhibit specificity for microsporidian MetAP2.

Structural and functional characterization of a putative polysaccharide deacetylase of the human parasite Encephalitozoon cuniculi.

The microsporidian Encephalitozoon cuniculi is an intracellular eukaryotic parasite considered to be an emerging opportunistic human pathogen. The infectious stage of this parasite is a unicellular spore that is surrounded by a chitin containing endospore layer and an external proteinaceous exospore. A putative chitin deacetylase (ECU11_0510) localizes to the interface between the plasma membrane and the endospore. Chitin deacetylases are family 4 carbohydrate esterases in the CAZY classification, and several bacterial members of this family are involved in evading lysis by host glycosidases, through partial de-N-acetylation of cell wall peptidoglycan. Similarly, ECU11_0510 could be important for E. cuniculi survival in the host, by protecting the chitin layer from hydrolysis by human chitinases. Here, we describe the biochemical, structural, and glycan binding properties of the protein. Enzymatic analyses showed that the putative deacetylase is unable to deacetylate chitooligosaccharides or crystalline beta-chitin. Furthermore, carbohydrate microarray analysis revealed that the protein bound neither chitooligosaccharides nor any of a wide range of other glycans or chitin. The high resolution crystal structure revealed dramatic rearrangements in the positions of catalytic and substrate binding residues, which explain the loss of deacetylase activity, adding to the unusual structural plasticity observed in other members of this esterase family. Thus, it appears that the ECU11_0510 protein is not a carbohydrate deacetylase and may fulfill an as yet undiscovered role in the E. cuniculi parasite.

Stripped-down DNA repair in a highly reduced parasite.

BACKGROUND: Encephalitozoon cuniculi is a member of a distinctive group of single-celled parasitic eukaryotes called microsporidia, which are closely related to fungi. Some of these organisms, including E. cuniculi, also have uniquely small genomes that are within the prokaryotic range. Thus, E. cuniculi has undergone a massive genome reduction which has resulted in a loss of genes from diverse biological pathways, including those that act in DNA repair.DNA repair is essential to any living cell. A loss of these mechanisms invariably results in accumulation of mutations and/or cell death. Six major pathways of DNA repair in eukaryotes include: non-homologous end joining (NHEJ), homologous recombination repair (HRR), mismatch repair (MMR), nucleotide excision repair (NER), base excision repair (BER) and methyltransferase repair. DNA polymerases are also critical players in DNA repair processes. Given the close relationship between microsporidia and fungi, the repair mechanisms present in E. cuniculi were compared to those of the yeast Saccharomyces cerevisiae to ascertain how the process of genome reduction has affected the DNA repair pathways. RESULTS: E. cuniculi lacks 16 (plus another 6 potential absences) of the 56 DNA repair genes sought via BLASTP and PSI-BLAST searches. Six of 14 DNA polymerases or polymerase subunits are also absent in E. cuniculi. All of these genes are relatively well conserved within eukaryotes. The absence of genes is not distributed equally among the different repair pathways; some pathways lack only one protein, while there is a striking absence of many proteins that are components of both double strand break repair pathways. All specialized repair polymerases are also absent. CONCLUSION: Given the large number of DNA repair genes that are absent from the double strand break repair pathways, E. cuniculi is a prime candidate for the study of double strand break repair with minimal machinery. Strikingly, all of the double strand break repair genes that have been retained by E. cuniculi participate in other biological pathways.

System for expression of microsporidian methionine amino peptidase type 2 (MetAP2) in the yeast Saccharomyces cerevisiae.

Microsporidia are parasitic protists of all classes of vertebrates and most invertebrates. They recently emerged as important infections in various immunosuppressed and immunocompetent patient populations. They are also important veterinary and agricultural pathogens. Current therapies for microsporidiosis include benzimidazoles, which bind tubulin-inhibiting microtubule assembly, and fumagillin and its derivatives, which bind and inhibit methionine amino peptidase type 2 (MetAP2). Benzimidazoles are not active against Enterocytozoon bieneusi, the most common cause of human microsporidiosis. Fumagillin is active against most microsporidia, including E. bieneusi, but thrombocytopenia has been a problem in clinical trials. There is a pressing need for more-specific microsporidian MetAP2 inhibitors. To expedite and facilitate the discovery of safe and effective MetAP2 inhibitors, we have engineered Saccharomyces cerevisiae to be dependent on Encephalitozoon cuniculi MetAP2 (EcMetAP2) for its growth, where EcMetAP2 is harbored on an episomal uracil-selectable tetracycline-regulated plasmid. We have also constructed a leucine-selectable tetracycline-regulated expression plasmid into which any MetAP2 gene can be cloned. By utilizing a 5-fluoroorotic acid-mediated plasmid shuffle in the EcMetAP2 yeast strain, a yeast strain can be generated whose growth is dependent on MetAP2 from any organism. The level of heterologous MetAP2 gene expression can be controlled by the addition of tetracycline to the growth medium. These yeast strains should permit high-throughput screening for the identification of new inhibitors with high specificity and activity toward microsporidian MetAP2.

Proteolytic activity in Encephalitozoon cuniculi sporogonial stages: predominance of metallopeptidases including an aminopeptidase-P-like enzyme.

A fraction enriched in spore precursor cells (sporoblasts) of the microsporidian Encephalitozoon cuniculi, an intracellular parasite of mammals, was obtained by Percoll gradient centrifugation. Soluble extracts of these cells exhibited proteolytic activity towards azocasein, with an alkaline optimum pH range (9-10). Prevalence of some metallopeptidases was supported by the stimulating effect of Ca2+, Mg2+, Mn2+ and Zn2+ ions, and inhibition by two chelating agents (EDTA and 1,10-phenanthroline), a thiol reductant (dithiothreitol) and two aminopeptidase inhibitors (bestatin and apstatin). Zymographic analysis revealed four caseinolytic bands at about 76, 70, 55 and 50 kDa. Mass spectrometry of tryptic peptides from one-dimensional gel slices identified a cytosol (leucine) aminopeptidase homologue (M17 family) in 50-kDa band and an enzyme similar to aminopeptidase P (AP-P) of cytosolic type (M24B subfamily) in 70-kDa band. Multiple sequence alignments showed conservation of critical residues for catalysis and metal binding. A long insertion in a common position was found in AP-P sequences from E. cuniculi and Nosema locustae, an insect-infecting microsporidian. The expression of cytosolic AP-P in sporogonial stages of microsporidia may suggest a key role in the attack of proline-containing peptides as a prerequisite to long-duration biosynthesis of structural proteins destined to the sporal polar tube.

The putative chitin deacetylase of Encephalitozoon cuniculi: a surface protein implicated in microsporidian spore-wall formation.

Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parasites. They differentiate into resistant spores that are protected by a thick cell wall composed of glycoproteins and chitin. Despite an extensive description of the fibrillar structure of this wall, very little is known about its protein components and deposit mechanisms. In this study on the human pathogen Encephalitozoon cuniculi, we identify by mass spectrometry the target of polyclonal antibodies previously raised against a 33-kDa protein located at the outer face of the parasite plasma membrane. This 254-amino acid protein is encoded by the ECU11_0510 open reading frame and presents two isoforms of 33 and 55 kDa. Sequence analysis supports an assignment to the polysaccharide deacetylase family with a suspected chitin deacetylase activity (EcCDA). As demonstrated by TEM studies, EcCDA is present at the plasma membrane of the early stages of E. cuniculi life-cycle. At the sporoblast stage, the enzyme accumulates especially in paramural bodies which are convolutions of the plasma membrane opened to the wall. The identification of an EcCDA homologue in the insect parasite Antonospora locustae (ex Nosema locustae) suggests a widespread distribution of this enzyme among Microsporidia. This characterization of a new microsporidian surface protein creates new perspectives to understand spore wall formation and spore resistance.

Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis.

The Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase Ecm1 has been characterized structurally but not biochemically. Here we show that purified Ecm1 is a monomeric protein that catalyzes methyl transfer from S-adenosylmethionine (AdoMet) to GTP. The reaction is cofactor-independent and optimal at pH 7.5. Ecm1 also methylates GpppA, GDP, and dGTP but not ATP, CTP, UTP, ITP, or m(7)GTP. The affinity of Ecm1 for the cap dinucleotide GpppA (K 0.1 mm) is higher than that for GTP (K(m) 1 mm) or GDP (K(m) 2.4 mm). Methylation of GTP by Ecm1 in the presence of 5 microm AdoMet is inhibited by the reaction product AdoHcy (IC(50) 4 microm) and by substrate analogs sinefungin (IC(50) 1.5 microm), aza-AdoMet (IC(50) 100 microm), and carbocyclic aza-AdoMet (IC(50) 35 microm). The crystal structure of an Ecm1.aza-AdoMet binary complex reveals that the inhibitor occupies the same site as AdoMet. Structure-function analysis of Ecm1 by alanine scanning and conservative substitutions identified functional groups necessary for methyltransferase activity in vivo. Amino acids Lys-54, Asp-70, Asp-78, and Asp-94, which comprise the AdoMet-binding site, and Phe-141, which contacts the cap guanosine, are essential for cap methyltransferase activity in vitro.

Phylogenetic positions of several amitochondriate protozoa--evidence from phylogenetic analysis of DNA topoisomerase II.

Several groups of parasitic protozoa, as represented by Giardia, Trichomonas, Entamoeba and Microsporida, were once widely considered to be the most primitive extant eukaryotic group--Archezoa. The main evidence for this is their 'lacking mitochondria' and possessing some other primitive features between prokaryotes and eukaryotes, and being basal to all eukaryotes with mitochondria in phylogenies inferred from many molecules. Some authors even proposed that these organisms diverged before the endosymbiotic origin of mitochondria within eukaryotes. This view was once considered to be very significant to the study of origin and evolution of eukaryotic cells (eukaryotes). However, in recent years this has been challenged by accumulating evidence from new studies. Here the sequences of DNA topoisomerase II in G lamblia, T. vaginalis and E. histolytica were identified first by PCR and sequencing, then combining with the sequence data of the microsporidia Encephalitozoon cunicul and other eukaryotic groups of different evolutionary positions from GenBank, phylogenetic trees were constructed by various methods to investigate the evolutionary positions of these amitochondriate protozoa. Our results showed that since the characteristics of DNA topoisomerase II make it avoid the defect of 'long-branch attraction' appearing in the previous phylogenetic analyses, our trees can not only reflect effectively the relationship of different major eukaryotic groups, which is widely accepted, but also reveal phylogenetic positions for these amitochondriate protozoa, which is different from the previous phylogenetic trees. They are not the earliest-branching eukaryotes, but diverged after some mitochondriate organisms such as kinetoplastids and mycetozoan; they are not a united group but occupy different phylogenetic positions. Combining with the recent cytological findings of mitochondria-like organelles in them, we think that though some of them (e.g. diplomonads, as represented by Giardia) may occupy a very low evolutionary position, generally these organisms are not as extremely primitive as was thought before; they should be polyphyletic groups diverging after the endosymbiotic origin of mitochondrion to adapt themselves to anaerobic parasitic life.

The genes encoding cAMP-dependent protein kinase catalytic subunit homologues of the microsporidia Encephalitozoon intestinalis and E. cuniculi: molecular characterisation and phylogenetic analysis.

A gene encoding a protein kinase was identified by homology-based PCR amplification in Encephalitozoon intestinalis, a microsporidian parasite pathogenic to humans, and its orthologue has been identified by database mining in the genome of the related species E. cuniculi, whose sequence has been recently published. Phylogenetic analysis revealed that the proteins encoded by these genes are homologues of the cAMP-dependent protein kinase catalytic subunits (PKAc). Southern blot analysis indicated that the EiPKAc gene is present in two copies in the E. intestinalis genome, whereas the E. cuniculi orthologue (EcPKAc) is a single copy gene. RT-PCR data showed that the EiPKAc gene is expressed in at least one of the intracellular stages during infection of the mammalian host cell by E. intestinalis.

An encephalitozoon cuniculi ortholog of the RNA polymerase II carboxyl-terminal domain (CTD) serine phosphatase Fcp1.

Fcp1 is an essential protein serine phosphatase that dephosphorylates Ser2 or Ser5 of the RNA polymerase II carboxyl-terminal domain (CTD) heptad repeat Y(1)S(2)P(3)T(4)S(5)P(6)S(7). The CTD of the microsporidian parasite Encephalitozoon cuniculi consists of 15 heptad repeats, which approximates the minimal CTD length requirement for cell viability in yeast. Here we show that E. cuniculi encodes a minimized 411-aa Fcp1-like protein (EcFcp1), which consists of a DxDx(T/V) phosphatase domain and a BRCA1 carboxyl terminus (BRCT) domain but lacks the large N- and C-terminal domains found in fungal and metazoan Fcp1 enzymes. Nonetheless, EcFcp1 can function in lieu of Saccharomyces cerevisiae Fcp1 to sustain yeast cell growth. Recombinant EcFcp1 is a monomeric enzyme with intrinsic phosphatase activity against nonspecific (p-nitrophenyl phosphate) and specific (CTD-PO(4)) substrates. EcFcp1 dephosphorylates CTD positions Ser2 and Ser5 with similar efficacy in vitro. We exploit synthetic CTD Ser2-PO(4) and Ser5-PO(4) peptides to define minimized substrates for EcFcp1 and to illuminate the importance of CTD primary structure in Ser2 and Ser5 phosphatase activity.

A comparison of homologous genes encoding aminopeptidases among bird and human Encephalitozoon hellem isolates and a rabbit E. cuniculi isolate.

Encephalitozoon cuniculi and E. hellem are often recognized as the agents of human microsporidiosis, but less than optimal therapy is available for treatment. The identification of enzymes critical to the parasitic life cycle is an important step in finding targets for potential drug development. Aminopeptidase gene sequences were obtained from cDNA and gDNA from avian and human E. hellem isolates and from a rabbit E. cuniculi isolate. At the amino acid level, the aminopeptidase sequences from the E. hellem human and bird isolates share >99% identity and are nearly 70% identical with the E. cuniculi sequence. Conserved HEXXH and GAMEN motifs classify the predicted aminopeptidase in the MA clan of the M1 family. The obtained aminopeptidase gene sequences are likely homologous to the previously reported E. cuniculi glutamyl aminopeptidase. The conservation of this aminopeptidase between species and divergence from mammalian aminopeptidases indicate that this enzyme may be a valid target for drug therapy.

Polyamine metabolism in a member of the phylum Microspora (Encephalitozoon cuniculi): effects of polyamine analogues.

The uptake, biosynthesis and catabolism of polyamines in the microsporidian parasite Encephalitozoon cuniculi are detailed with reference to the effects of oligoamine and arylamine analogues of polyamines. Enc. cuniculi, an intracellular parasite of mammalian cells, has both biosynthetic and catabolic enzymes of polyamine metabolism, as demonstrated in cell-free extracts of mature spores. The uptake of polyamines was measured in immature, pre-emergent spores isolated from host cells by Percoll gradient. Spermine was rapidly taken up and metabolized to spermidine and an unknown, possibly acetamidopropanal, by spermidine/spermine N(1)-acetyltransferase (SSAT) and polyamine oxidase (PAO). Most of the spermidine and the unknown product were found in the cell incubation medium, indicating they were released from the cell. bis(Ethyl) oligoamine analogues of polyamines, such as SL-11144 and SL-11158, as well as arylamine analogues [BW-1, a bis(phenylbenzyl) 3-7-3 analogue] blocked uptake and interconversion of spermine at micromolar levels and, in the case of BW-1, acted as substrate for PAO. The Enc. cuniculi PAO activity differed from that found in mammalian cells with respect to pH optimum, substrate specificity and sensitivity to known PAO inhibitors. SL-11158 inhibited SSAT activity with a mixed type of inhibition in which the analogue had a 70-fold higher affinity for the enzyme than the natural substrate, spermine. The interest in Enc. cuniculi polyamine metabolism and the biochemical effects of these polyamine analogues is warranted since they cure model infections of Enc. cuniculi in mice and are potential candidates for human clinical trials.