High-resolution epitope mapping by HX MS reveals the pathogenic mechanism and a possible therapy for autoimmune TTP syndrome.

Acquired thrombotic thrombocytopenic purpura (TTP), a thrombotic disorder that is fatal in almost all cases if not treated promptly, is primarily caused by IgG-type autoantibodies that inhibit the ability of the ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) metalloprotease to cleave von Willebrand factor (VWF). Because the mechanism of autoantibody-mediated inhibition of ADAMTS13 activity is not known, the only effective therapy so far is repeated whole-body plasma exchange. We used hydrogen-deuterium exchange mass spectrometry (HX MS) to determine the ADAMTS13 binding epitope for three representative human monoclonal autoantibodies, isolated from TTP patients by phage display as tethered single-chain fragments of the variable regions (scFvs). All three scFvs bind the same conformationally discontinuous epitopic region on five small solvent-exposed loops in the spacer domain of ADAMTS13. The same epitopic region is also bound by most polyclonal IgG autoantibodies in 23 TTP patients that we tested. The ability of ADAMTS13 to proteolyze VWF is impaired by the binding of autoantibodies at the epitopic loops in the spacer domain, by the deletion of individual epitopic loops, and by some local mutations. Structural considerations and HX MS results rule out any disruptive structure change effect in the distant ADAMTS13 metalloprotease domain. Instead, it appears that the same ADAMTS13 loop segments that bind the autoantibodies are also responsible for correct binding to the VWF substrate. If so, the autoantibodies must prevent VWF proteolysis simply by physically blocking normal ADAMTS13 to VWF interaction. These results point to the mechanism for autoantibody action and an avenue for therapeutic intervention.

L1 retrotransposition occurs mainly in embryogenesis and creates somatic mosaicism.

Long Interspersed Element 1 (L1) is a retrotransposon that comprises approximately 17% of the human genome. Despite its abundance in mammalian genomes, relatively little is understood about L1 retrotransposition in vivo. To study the timing and tissue specificity of retrotransposition, we created transgenic mouse and rat models containing human or mouse L1 elements controlled by their endogenous promoters. Here, we demonstrate abundant L1 RNA in both germ cells and embryos. However, the integration events usually occur in embryogenesis rather than in germ cells and are not heritable. We further demonstrate L1 RNA in preimplantation embryos lacking the L1 transgene and L1 somatic retrotransposition events in blastocysts and adults lacking the transgene. Together, these data indicate that L1 RNA transcribed in male or female germ cells can be carried over through fertilization and integrate during embryogenesis, an interesting example of heritability of RNA independent of its encoding DNA. Thus, L1 creates somatic mosaicism during mammalian development, suggesting a role for L1 in carcinogenesis and other disease.

ADAMTS13 autoantibodies cloned from patients with acquired thrombotic thrombocytopenic purpura: 1. Structural and functional characterization in vitro.

BACKGROUND: Acquired thrombotic thrombocytopenia purpura (TTP) is a life-threatening illness caused by autoantibodies that decrease the activity of ADAMTS13, the von Willebrand factor-cleaving protease. Despite efficacy of plasma exchange, mortality remains high and relapse is common. Improved therapies may come from understanding the diversity of pathogenic autoantibodies on a molecular or genetic level. Cloning comprehensive repertoires of patient autoantibodies can provide the necessary tools for studying immunobiology of disease and developing animal models. STUDY DESIGN AND METHODS: Anti-ADAMTS13 antibodies were cloned from four patients with acquired TTP using phage display and characterized with respect to genetic origin, inhibition of ADAMTS13 proteolytic activity, and epitope specificity. Anti-idiotypic antisera raised to a subset of autoantibodies enabled comparison of their relatedness to each other and to polyclonal immunoglobulin (Ig)G in patient plasma. RESULTS: Fifty-one unique antibodies were isolated comprising epitope specificities resembling the diversity found in circulating patient IgG. Antibodies directed both to the amino terminal domains and to those requiring the ADAMTS13 cysteine-rich/spacer region for binding inhibited proteolytic activity, while those solely targeting carboxy-terminal domains were noninhibitory. Anti-idiotypic antisera raised to a subset of antibody clones crossreacted with and reduced the inhibitory activity of polyclonal IgG from a set of unrelated patients. CONCLUSIONS: Anti-ADAMTS13 autoantibodies isolated by repertoire cloning display the diversity of epitope specificities found in patient plasma and provide tools for developing animal models of acquired TTP. Shared idiotypes of inhibitory clones with circulating IgG from multiple patients suggest common features of pathogenic autoantibodies that could be exploited for developing more targeted therapies.

ADAMTS13 autoantibodies cloned from patients with acquired thrombotic thrombocytopenic purpura: 2. Pathogenicity in an animal model.

BACKGROUND: Acquired thrombotic thrombocytopenic purpura (TTP) is a potentially fatal disease in which ultralarge von Willebrand factor (UL-VWF) multimers accumulate as a result of autoantibody inhibition of the VWF protease, ADAMTS13. Current treatment is not specifically directed at the responsible autoantibodies and in some cases is ineffective or of transient benefit. More rational, reliable, and durable therapies are needed, and a human autoantibody-mediated animal model would be useful for their development. Previously, TTP patient anti-ADAMTS13 single-chain variable-region fragments (scFv's) were cloned that inhibited ADAMTS13 proteolytic activity in vitro and expressed features in common with inhibitory immunoglobulin G in patient plasma. Here, pathogenicity of these scFv's is explored in vivo by transfecting mice with inhibitory antibody cDNA. STUDY DESIGN AND METHODS: Hydrodynamic tail vein injection of naked DNA encoding human anti-ADAMTS13 scFv was used to create sustained ADAMTS13 inhibition in mice. Accumulation of UL-VWF multimers was measured and formation of platelet (PLT) thrombi after focal or systemic vascular injury was examined. RESULTS: Transfected mice expressed physiological plasma levels of human scFv and developed sustained ADAMTS13 inhibition and accumulation of unprocessed UL-VWF multimers. Induced focal endothelial injury generated PLT thrombi extending well beyond the site of initial injury, and systemic endothelial injury induced thrombocytopenia, schistocyte formation, PLT thrombi, and death. CONCLUSIONS: These results demonstrate for the first time the ability of human recombinant monovalent anti-ADAMTS13 antibody fragments to recapitulate key pathologic features of untreated acquired TTP in vivo, validating their clinical significance and providing an animal model for testing novel targeted therapeutic approaches.

Progressive Saturation Improves the Encapsulation of Functional Proteins in Nanoscale Polymer Vesicles.

PURPOSE: To develop a technique that maximizes the encapsulation of functional proteins within neutrally charged, fully PEGylated and nanoscale polymer vesicles (i.e., polymersomes). METHODS: Three conventional vesicle formation methods were utilized for encapsulation of myoglobin (Mb) in polymersomes of varying size, PEG length, and membrane thickness. Mb concentrations were monitored by UV-Vis spectroscopy, inductively coupled plasma optical emission spectroscopy (ICP-OES) and by the bicinchoninic acid (BCA) assay. Suspensions were subject to protease treatment to differentiate the amounts of surface-associated vs. encapsulated Mb. Polymersome sizes and morphologies were monitored by dynamic light scattering (DLS) and by cryogenic transmission electron microscopy (cryo-TEM), respectively. Binding and release of oxygen were measured using a Hemeox analyzer. RESULTS: Using the established "thin-film rehydration" and "direct hydration" methods, Mb was found to be largely surface-associated with negligible aqueous encapsulation within polymersome suspensions. Through iterative optimization, a novel "progressive saturation" technique was developed that greatly increased the final concentrations of Mb (from < 0.5 to > 2.0 mg/mL in solution), the final weight ratio of Mb-to-polymer that could be reproducibly obtained (from < 1 to > 4 w/w% Mb/polymer), as well as the overall efficiency of Mb encapsulation (from < 5 to > 90%). Stable vesicle morphologies were verified by cryo-TEM; the suspensions also displayed no signs of aggregate formation for > 2 weeks as assessed by DLS. "Progressive saturation" was further utilized for the encapsulation of a variety of other proteins, ranging in size from 17 to 450 kDa. CONCLUSIONS: Compared to established vesicle formation methods, "progressive saturation" increases the quantities of functional proteins that may be encapsulated in nanoscale polymersomes.

A mouse model of human L1 retrotransposition.

The L1 retrotransposon has had an immense impact on the size and structure of the human genome through a variety of mechanisms, including insertional mutagenesis. To study retrotransposition in a living organism, we created a mouse model of human L1 retrotransposition. Here we show that L1 elements can retrotranspose in male germ cells, and that expression of a human L1 element under the control of its endogenous promoter is restricted to testis and ovary. In the mouse line with the highest level of L1 expression, we found two de novo L1 insertions in 135 offspring. Both insertions were structurally indistinguishable from natural endogenous insertions. This suggests that an individual L1 element can have substantial mutagenic potential. In addition to providing a valuable in vivo model of retrotransposition in mammals, these mice are an important step in the development of a new random mutagenesis system.

Erratum: Cas-CLOVER is a novel high-fidelity nuclease for safe and robust generation of TSCM-enriched allogeneic CAR-T cells.

[This corrects the article DOI: 10.1016/j.omtn.2022.06.003.].

Cas-CLOVER is a novel high-fidelity nuclease for safe and robust generation of TSCM-enriched allogeneic CAR-T cells.

The use of T cells from healthy donors for allogeneic chimeric antigen receptor T (CAR-T) cell cancer therapy is attractive because healthy donor T cells can produce versatile off-the-shelf CAR-T treatments. To maximize safety and durability of allogeneic products, the endogenous T cell receptor and major histocompatibility complex class I molecules are often removed via knockout of T cell receptor beta constant (TRBC) (or T cell receptor alpha constant [TRAC]) and B2M, respectively. However, gene editing tools (e.g., CRISPR-Cas9) can display poor fidelity, which may result in dangerous off-target mutations. Additionally, many gene editing technologies require T cell activation, resulting in a low percentage of desirable stem cell memory T cells (TSCM). We characterize an RNA-guided endonuclease, called Cas-CLOVER, consisting of the Clo051 nuclease domain fused with catalytically dead Cas9. In primary T cells from multiple donors, we find that Cas-CLOVER is a high-fidelity site-specific nuclease, with low off-target activity. Notably, Cas-CLOVER yields efficient multiplexed gene editing in resting T cells. In conjunction with the piggyBac transposon for delivery of a CAR transgene against the B cell maturation antigen (BCMA), we produce allogeneic CAR-T cells composed of high percentages of TSCM cells and possessing potent in vivo anti-tumor cytotoxicity.

The SRG rat, a Sprague-Dawley Rag2/Il2rg double-knockout validated for human tumor oncology studies.

We have created the immunodeficient SRG rat, a Sprague-Dawley Rag2/Il2rg double knockout that lacks mature B cells, T cells, and circulating NK cells. This model has been tested and validated for use in oncology (SRG OncoRat®). The SRG rat demonstrates efficient tumor take rates and growth kinetics with different human cancer cell lines and PDXs. Although multiple immunodeficient rodent strains are available, some important human cancer cell lines exhibit poor tumor growth and high variability in those models. The VCaP prostate cancer model is one such cell line that engrafts unreliably and grows irregularly in existing models but displays over 90% engraftment rate in the SRG rat with uniform growth kinetics. Since rats can support much larger tumors than mice, the SRG rat is an attractive host for PDX establishment. Surgically resected NSCLC tissue from nine patients were implanted in SRG rats, seven of which engrafted and grew for an overall success rate of 78%. These developed into a large tumor volume, over 20,000 mm3 in the first passage, which would provide an ample source of tissue for characterization and/or subsequent passage into NSG mice for drug efficacy studies. Molecular characterization and histological analyses were performed for three PDX lines and showed high concordance between passages 1, 2 and 3 (P1, P2, P3), and the original patient sample. Our data suggest the SRG OncoRat is a valuable tool for establishing PDX banks and thus serves as an alternative to current PDX mouse models hindered by low engraftment rates, slow tumor growth kinetics, and multiple passages to develop adequate tissue banks.

Sprague Dawley Rag2-Null Rats Created from Engineered Spermatogonial Stem Cells Are Immunodeficient and Permissive to Human Xenografts.

The rat is the preferred model for toxicology studies, and it offers distinctive advantages over the mouse as a preclinical research model including larger sample size collection, lower rates of drug clearance, and relative ease of surgical manipulation. An immunodeficient rat would allow for larger tumor size development, prolonged dosing and drug efficacy studies, and preliminary toxicologic testing and pharmacokinetic/pharmacodynamic studies in the same model animal. Here, we created an immunodeficient rat with a functional deletion of the Recombination Activating Gene 2 (Rag2) gene, using genetically modified spermatogonial stem cells (SSC). We targeted the Rag2 gene in rat SSCs with TALENs and transplanted these Rag2-deficient SSCs into sterile recipients. Offspring were genotyped, and a founder with a 27 bp deletion mutation was identified and bred to homozygosity to produce the Sprague-Dawley Rag2 - Rag2 tm1Hera (SDR) knockout rat. We demonstrated that SDR rat lacks mature B and T cells. Furthermore, the SDR rat model was permissive to growth of human glioblastoma cell line subcutaneously resulting in successful growth of tumors. In addition, a human KRAS-mutant non-small cell lung cancer cell line (H358), a patient-derived high-grade serous ovarian cancer cell line (OV81), and a patient-derived recurrent endometrial cancer cell line (OV185) were transplanted subcutaneously to test the ability of the SDR rat to accommodate human xenografts from multiple tissue types. All human cancer cell lines showed efficient tumor uptake and growth kinetics indicating that the SDR rat is a viable host for a range of xenograft studies. Mol Cancer Ther; 17(11); 2481-9. ©2018 AACR.

Loss of the trpc4 gene is associated with a reduction in cocaine self-administration and reduced spontaneous ventral tegmental area dopamine neuronal activity, without deficits in learning for natural rewards.

Among the canonical transient receptor potential (TRPC) channels, the TRPC4 non-selective cation channel is one of the most abundantly expressed subtypes within mammalian corticolimbic brain regions, but its functional and behavioral role is unknown. To identify a function for TRPC4 channels we compared the performance of rats with a genetic knockout of the trpc4 gene (trpc4 KO) to wild-type (WT) controls on the acquisition of simple and complex learning for natural rewards, and on cocaine self-administration (SA). Despite the abundant distribution of TRPC4 channels through the corticolimbic brain regions, we found trpc4 KO rats exhibited normal learning in Y-maze and complex reversal shift paradigms. However, a deficit was observed in cocaine SA in the trpc4 KO group, which infused significantly less cocaine than WT controls despite displaying normal sucrose SA. Given the important role of ventral tegmental area (VTA) dopamine neurons in cocaine SA, we hypothesized that TRPC4 channels may regulate basal dopamine neuron excitability. Double-immunolabeling showed a selective expression of TRPC4 channels in a subpopulation of putative dopamine neurons in the VTA. Ex vivo recordings of spontaneous VTA dopamine neuronal activity from acute brain slices revealed fewer cells with high-frequency firing rates in trpc4 KO rats compared to WT controls. Since deletion of the trpc4 gene does not impair learning involving natural rewards, but reduces cocaine SA, these data demonstrate a potentially novel role for TRPC4 channels in dopamine systems and may offer a new pharmacological target for more effective treatment of a variety of dopamine disorders.

Myosin Id is required for planar cell polarity in ciliated tracheal and ependymal epithelial cells.

In wild type (WT) tracheal epithelial cells, ciliary basal bodies are oriented such that all cilia on the cell surface beat in the same upward direction. This precise alignment of basal bodies and, as a result, the ciliary axoneme, is termed rotational planar cell polarity (PCP). Rotational PCP in the multi-ciliated epithelial cells of the trachea is perturbed in rats lacking myosin Id (Myo1d). Myo1d is localized in the F-actin and basal body rich subapical cortex of the ciliated tracheal epithelial cell. Scanning and transmission electron microscopy of Myo1d knock out (KO) trachea revealed that the unidirectional bending pattern is disrupted. Instead, cilia splay out in a disordered, often radial pattern. Measurement of the alignment axis of the central pair axonemal microtubules was much more variable in the KO, another indicator that rotational PCP is perturbed. The asymmetric localization of the PCP core protein Vangl1 is lost. Both the velocity and linearity of cilia-driven movement of beads above the tracheal mucosal surface was impaired in the Myo1d KO. Multi-ciliated brain ependymal epithelial cells exhibit a second form of PCP termed translational PCP in which basal bodies and attached cilia are clustered at the anterior side of the cell. The precise asymmetric clustering of cilia is disrupted in the ependymal cells of the Myo1d KO rat. While basal body clustering is maintained, left-right positioning of the clusters is lost.

TMEM16C facilitates Na(+)-activated K+ currents in rat sensory neurons and regulates pain processing.

TMEM16C belongs to the TMEM16 family, which includes the Ca(2+)-activated Cl(-) channels TMEM16A and TMEM16B and a small-conductance, Ca(2+)-activated, nonselective cation channel (SCAN), TMEM16F. We found that in rat dorsal root ganglia (DRG) TMEM16C was expressed mainly in the IB4-positive, non-peptidergic nociceptors that also express the sodium-activated potassium (K(Na)) channel Slack. Together these channel proteins promote K(Na) channel activity and dampen neuronal excitability. DRG from TMEM16C knockout rats had diminished Slack expression, broadened action potentials and increased excitability. Moreover, the TMEM16C knockout rats, as well as rats with Slack knockdown by intrathecal injection of short interfering RNA, exhibited increased thermal and mechanical sensitivity. Experiments involving heterologous expression in HEK293 cells further showed that TMEM16C modulated the single-channel activity of Slack channels and increased its sodium sensitivity. Our study thus reveals that TMEM16C enhances K(Na) channel activity in DRG neurons and regulates the processing of pain messages.

Juvenile cataract-associated mutation of solute carrier SLC16A12 impairs trafficking of the protein to the plasma membrane.

PURPOSE: SLC16A12 encodes an orphan member of the monocarboxylate transporter family, MCT12. A nonsense mutation in SLC16A12 (c.643C>T; p.Q215X) causes juvenile cataract with a dominant inheritance pattern. In the present study, in vitro and in vivo experimental models were used to gain insight into how the SLC16A12 (c.643C>T) mutation leads to cataract formation. METHODS: MCT12 peptide antibodies were generated and used to examine the expression of MCT12 in the lens using immuno-confocal microscopy. To determine whether loss of Slc16a12 resulted in cataract formation, a Slc16a12 hypomorphic rat generated by transposon insertional mutagenesis was characterized using RT-PCR, slit lamp microscopy and histologic methods. Exogenous expression of MCT12 and MCT12:214Δ, a mimic of the mutant allele, were used to assess protein expression and trafficking. RESULTS: MCT12 protein was detected in the lens epithelium and secondary fiber cells at postnatal day 1. In the Slc16a12(TKO) rat, complete loss of MCT12 did not result in any detectable ocular phenotype. Exogenous expression of MCT12-GFP and MCT12:214Δ-GFP revealed that the full-length protein was trafficked to the plasma membrane (PM), whereas the truncated protein was retained in the endoplasmic reticulum (ER). When both MCT12 and MCT12:214Δ were coexpressed, to mimic the heterozygous patient genotype, the truncated protein was retained in the ER whereas full-length MCT12 was trafficked to the PM. Furthermore, MCT12 was identified as another MCT isoform that requires CD147 for trafficking to the cell surface. CONCLUSIONS: These data support a model whereby the SLC16A12 (c.643C>T) mutation causes juvenile cataract by a defect in protein trafficking rather than by haploinsufficiency.

Exacerbated pulmonary arterial hypertension and right ventricular hypertrophy in animals with loss of function of extracellular superoxide dismutase.

Studies have demonstrated that increased oxidative stress contributes to the pathogenesis and the development of pulmonary artery hypertension (PAH). Extracellular superoxide dismutase (SOD3) is essential for removing extracellular superoxide anions, and it is highly expressed in lung tissue. However, it is not clear whether endogenous SOD3 can influence the development of PAH. Here we examined the effect of SOD3 knockout on hypoxia-induced PAH in mice and a loss-of-function SOD3 gene mutation (SOD3(E124D)) on monocrotaline (40 mg/kg)-induced PAH in rats. SOD3 knockout significantly exacerbated 2 weeks of hypoxia-induced right ventricular (RV) pressure and RV hypertrophy, whereas RV pressure in SOD3 knockout mice under normoxic conditions is similar to wild-type controls. In untreated control rats at age of 8 weeks, there was no significant difference between wild-type and SOD3(E124D) rats in RV pressure and the ratio of RV weight:left ventricular weight (0.25±0.02 in wild-type rats versus 0.25±0.01 in SOD3(E124D) rats). However, monocrotaline caused significantly greater increases of RV pressure in SOD3(E124D) rats (48.6±1.8 mm Hg in wild-type versus 57.5±3.1 mm Hg in SOD3(E124D) rats), of the ratio of RV weight:left ventricular weight (0.41±0.01 versus 0.50±0.09; P<0.05), and of the percentage of fully muscularized small arterioles in SOD3(E124D) rats (55.2±2.3% versus 69.9±2.6%; P<0.05). Together, these findings indicate that the endogenous SOD3 has no role in the development of PAH under control conditions but plays an important role in protecting the lung from the development of PAH under stress conditions.

Mutagenesis in rodents using the L1 retrotransposon.

LINE1 (L1) retrotransposons are genetic elements that are present in all mammalian genomes. L1s are active in both humans and mice, and are capable of copying themselves and inserting the copy into a new genomic location. These de novo insertions occasionally result in disease. Endogenous L1 retrotransposons can be modified to increase their activity and mutagenic power in a variety of ways. Here we outline the advantages of using modified L1 retrotransposons for performing random mutagenesis in rodents and discuss several potential applications.

A novel testis ubiquitin-binding protein gene arose by exon shuffling in hominoids.

Most new genes arise by duplication of existing gene structures, after which relaxed selection on the new copy frequently leads to mutational inactivation of the duplicate; only rarely will a new gene with modified function emerge. Here we describe a unique mechanism of gene creation, whereby new combinations of functional domains are assembled at the RNA level from distinct genes, and the resulting chimera is then reverse transcribed and integrated into the genome by the L1 retrotransposon. We characterized a novel gene, which we termed PIP5K1A and PSMD4-like (PIPSL), created by this mechanism from an intergenic transcript between the phosphatidylinositol-4-phosphate 5-kinase (PIP5K1A) and the 26S proteasome subunit (PSMD4) genes in a hominoid ancestor. PIPSL is transcribed specifically in the testis both in humans and chimpanzees, and is post-transcriptionally repressed by independent mechanisms in these primate lineages. The PIPSL gene encodes a chimeric protein combining the lipid kinase domain of PIP5K1A and the ubiquitin-binding motifs of PSMD4. Strong positive selection on PIPSL led to its rapid divergence from the parental genes PIP5K1A and PSMD4, forming a chimeric protein with a distinct cellular localization and minimal lipid kinase activity, but significant affinity for cellular ubiquitinated proteins. PIPSL is a tightly regulated, testis-specific novel ubiquitin-binding protein formed by an unusual exon-shuffling mechanism in hominoid primates and represents a key example of rapid evolution of a testis-specific gene.

L1 integration in a transgenic mouse model.

To study integration of the human LINE-1 retrotransposon (L1) in vivo, we developed a transgenic mouse model of L1 retrotransposition that displays de novo somatic L1 insertions at a high frequency, occasionally several insertions per mouse. We mapped 3' integration sites of 51 insertions by Thermal Asymmetric Interlaced PCR (TAIL-PCR). Analysis of integration locations revealed a broad genomic distribution with a modest preference for intergenic regions. We characterized the complete structures of 33 de novo retrotransposition events. Our results highlight the large number of highly truncated L1s, as over 52% (27/51) of total integrants were <1/3 the length of a full-length element. New integrants carry all structural characteristics typical of genomic L1s, including a number with inversions, deletions, and 5'-end microhomologies to the target DNA sequence. Notably, at least 13% (7/51) of all insertions contain a short stretch of extra nucleotides at their 5' end, which we postulate result from template-jumping by the L1-encoded reverse transcriptase. We propose a unified model of L1 integration that explains all of the characteristic features of L1 retrotransposition, such as 5' truncations, inversions, extra nucleotide additions, and 5' boundary and inversion point microhomologies.

SVA elements are nonautonomous retrotransposons that cause disease in humans.

L1 elements are the only active autonomous retrotransposons in the human genome. The nonautonomous Alu elements, as well as processed pseudogenes, are retrotransposed by the L1 retrotransposition proteins working in trans. Here, we describe another repetitive sequence in the human genome, the SVA element. Our analysis reveals that SVA elements are currently active in the human genome. SVA elements, like Alus and L1s, occasionally insert into genes and cause disease. Furthermore, SVA elements are probably mobilized in trans by active L1 elements.