Uncovering the magnitude of African pangolin poaching with extensive nanopore DNA genotyping of seized scales.

Trade in pangolins is illegal, and yet tons of their scales and products are seized at various ports. These large seizures are challenging to process and comprehensively genotype for upstream provenance tracing and species identification for prosecution. We implemented a scalable DNA barcoding pipeline in which rapid DNA extraction and MinION sequencing were used to genotype a substantial proportion of pangolin scales subsampled from 2 record shipments seized in Singapore in 2019 (37.5 t). We used reference sequences to match the scales to phylogeographical regions of origin. In total, we identified 2346 cytochrome b (cytb) barcodes of white-bellied (Phataginus tricuspis) (from 1091 scales), black-bellied (Phataginus tetradactyla) (227 scales), and giant (Smutsia gigantea) (1028 scales) pangolins. Haplotype diversity was higher for P. tricuspis scales (121 haplotypes, 66 novel) than that for P. tetradactyla (22 haplotypes, 15 novel) and S. gigantea (25 haplotypes, 21 novel) scales. Of the novel haplotypes, 74.2% were likely from western and west-central Africa, suggesting potential resurgence of poaching and newly exploited populations in these regions. Our results illustrate the utility of extensively subsampling large seizures and outline an efficient molecular approach for rapid genetic screening that should be accessible to most forensic laboratories and enforcement agencies.

Revelación de la magnitud de la caza furtiva del pangolín africano mediante el genotipo extenso de nanoporos de ADN de escamas incautadas Resumen Aunque el mercado de pangolines es ilegal, se incautan toneladas de sus escamas y productos derivados en varios puertos comerciales. Es un reto procesar estas magnas incautaciones y obtener el genotipo completo para usarlo en la trazabilidad logística ascendente e identificación de la especie y así imponer sanciones. Implementamos una canalización escalable del código de barras de ADN en el cual usamos la extracción rápida de ADN y la secuenciación MinION para obtener el genotipo de una proporción sustancial de las escamas de pangolín submuestreadas en dos cargamentos incautados en 2019 en Singapur (37.5 t). Usamos secuencias referenciales para emparejar las escamas con las regiones filogeográficas de origen. Identificamos en total 2,346 códigos de citocromo b (cytb) del pangolín de vientre blanco (Phataginus tricuspis) (de 1,091 escamas), de vientre negro (P. tetradactyla) (227 escamas) y del pangolín gigante (Smutsia gigantea) (1,028 escamas). La diversidad de haplotipos fue mayor en las escamas de P. tricuspis (121 haplotipos, 66 nuevos) que en las de P. tetradactyla (22 haplotipos, 15 nuevos) y S. gigantea (25 haplotipos, 21 nuevos). De los haplotipos nuevos, el 74.2% probablemente provenía del occidente y centro­occidente de África, lo que sugiere un resurgimiento potencial de la caza furtiva y poblaciones recién explotadas en estas regiones. Nuestros resultados demuestran la utilidad de submuestrear extensivamente las grandes incautaciones y esboza una estrategia molecular eficiente para un análisis genético rápido que debería ser accesible para la mayoría de los laboratorios forenses y las autoridades de aplicación.

Detection of a novel Babesia sp. in Amblyomma javanense, an ectoparasite of Sunda pangolins.

BACKGROUND: Babesia is a protozoal, tick-borne parasite that can cause life-threatening disease in humans, wildlife and domestic animals worldwide. However, in Southeast Asia, little is known about the prevalence and diversity of Babesia species present in wildlife and the tick vectors responsible for its transmission. Recently, a novel Babesia species was reported in confiscated Sunda pangolins (Manis javanica) in Thailand. To investigate the presence of this parasite in Singapore, we conducted a molecular survey of Babesia spp. in free-roaming Sunda pangolins and their main ectoparasite, the Amblyomma javanense tick. METHODS: Ticks and tissue samples were opportunistically collected from live and dead Sunda pangolins and screened using a PCR assay targeting the 18S rRNA gene of Babesia spp. DNA barcoding of the cytochrome oxidase subunit I (COI) mitochondrial gene was used to confirm the species of ticks that were Babesia positive. RESULTS: A total of 296 ticks and 40 tissue samples were obtained from 21 Sunda pangolins throughout the 1-year study period. Babesia DNA was detected in five A. javanense ticks (minimum infection rate = 1.7%) and in nine different pangolins (52.9%) located across the country. Phylogenetic analysis revealed that the Babesia 18S sequences obtained from these samples grouped into a single monophyletic clade together with those derived from Sunda pangolins in Thailand and that this evolutionarily distinct species is basal to the Babesia sensu stricto clade, which encompasses a range of Babesia species that infect both domestic and wildlife vertebrate hosts. CONCLUSIONS: This is the first report documenting the detection of a Babesia species in A. javanense ticks, the main ectoparasite of Sunda pangolins. While our results showed that A. javanense can carry this novel Babesia sp., additional confirmatory studies are required to demonstrate vector competency. Further studies are also necessary to investigate the role of other transmission pathways given the low infection rate of ticks in relation to the high infection rate of Sunda pangolins. Although it appears that this novel Babesia sp. is of little to no pathogenicity to Sunda pangolins, its potential to cause disease in other animals or humans cannot be ruled out.

Telomerase recruitment to telomeres.

The ability of most cancer cells to grow indefinitely relies on the presence of functional telomerase to maintain telomeres, thus circumventing normal cellular senescence. A key feature of telomerase functionality is the localization of the enzyme complex to telomeres, a process which is highly regulated. A number of recent studies have reported data with significant implications for our understanding of telomerase recruitment to telomeres. A picture is emerging that this process is governed by a number of factors including telomeric binding proteins, structural features of the enzyme complex, cell cycle regulated processes such as DNA replication, and components of the DNA damage response pathway. In this review we summarize recent findings relating to this fundamental process in eukaryotes.

Alternative lengthening of telomeres in human cells.

Activation of a telomere maintenance mechanism appears to be essential for immortalization. In most human tumors and tumor cell lines, the telomere maintenance mechanism involves the activity of telomerase, a reverse transcriptase holoenzyme that synthesizes telomeric repeat DNA. In some cases, telomere maintenance occurs in the absence of telomerase activity by a mechanism referred to as alternative lengthening of telomeres (ALT). The development of telomere-targeted anticancer therapies will be facilitated by an understanding of the molecular mechanisms of ALT and of the means whereby ALT is repressed in normal cells.

Telomerase RNA bound by protein motifs specific to telomerase reverse transcriptase.

Telomerase reverse transcriptase (TERT) differs from many other reverse transcriptases in that it remains stably associated with its template-containing RNA subunit. Elements of TERT involved in binding the RNA subunit have now been identified by mutagenesis and in vitro reconstitution of the Tetrahymena ribonucleoprotein complex. Mutations in the reverse transcriptase motifs of TERT reduced activity as expected but did not greatly reduce its binding to the telomerase RNA. In contrast, all mutations in the T and CP motifs dramatically reduced RNA binding. We therefore suggest that the T and CP motifs of TERT function to hold on to the telomerase RNA, leaving the RNA template region free to translocate through the RT domain.

A mutant of Tetrahymena telomerase reverse transcriptase with increased processivity.

The protein catalytic subunit of telomerase (TERT) is a reverse transcriptase (RT) that utilizes an internal RNA molecule as a template for the extension of chromosomal DNA ends. In all retroviral RTs there is a conserved tyrosine two amino acids preceding the catalytic aspartic acids in motif C, a motif that is critical for catalysis. In TERTs, however, this position is a leucine, valine, or phenylalanine. We developed and characterized a robust in vitro reconstitution system for Tetrahymena telomerase and tested the effects of amino acid substitutions on activity. Substitution of the retroviral-like tyrosine in motif C did not change overall enzymatic activity but increased processivity. This increase in processivity correlated with an increased affinity for telomeric DNA primer. Substitution of an alanine did not increase processivity, while substitution of a phenylalanine had an intermediate effect. The data suggest that this amino acid is involved in interactions with the primer in telomerase as in other RTs, and show that mutating an amino acid to that conserved in retroviral RTs makes telomerase more closely resemble these other RTs.

Telomerase and the maintenance of chromosome ends.

The catalytic subunit of telomerase has recently been identified in diverse eukaryotes and shown to be a reverse transcriptase. Ectopic expression of this protein in normal human cells leads to lengthened telomeres and an extended in vitro life span. Other proteins that modulate telomerase activity in vivo are also being identified, including a functionally conserved family of proteins with Myb-like DNA-binding domains and proteins that are involved in DNA double-strand break repair.

Repression of an alternative mechanism for lengthening of telomeres in somatic cell hybrids.

Some immortalized cell lines maintain their telomeres in the absence of detectable telomerase activity by an alternative (ALT) mechanism. To study how telomere maintenance is controlled in ALT cells, we have fused an ALT cell line GM847 (SV40 immortalized human skin fibroblasts) with normal fibroblasts or with telomerase positive immortal human cell lines and have examined their proliferative potential and telomere dynamics. The telomeres in ALT cells are characteristically very heterogeneous in length, ranging from very short to very long. The ALT x normal hybrids underwent a rapid reduction in telomeric DNA and entered a senescence-like state. Immortal segregants rapidly reverted to the ALT telomere phenotype. Fusion of ALT cells to telomerase-positive immortal cells in the same immortalization complementation group resulted in hybrids that appeared immortal and also exhibited repression of the ALT telomere phenotype. In these hybrids, which were all telomerase-positive, we observed an initial rapid loss of most long telomeres, followed either by gradual loss of the remaining long telomeres at a rate similar to the rate of telomere shortening in normal telomerase-negative cells, or by maintenance of shortened telomeres. These data indicate the existence of a mechanism of rapid telomere deletion in human cells. They also demonstrate that normal cells and at least some telomerase-positive immortal cells contain repressors of the ALT telomere phenotype.

Telomerase reverse transcriptase genes identified in Tetrahymena thermophila and Oxytricha trifallax.

Telomerase reverse transcriptase (TERT) has been identified as the catalytic subunit of the chromosome end-replicating enzyme in Euplotes, yeasts, and mammals. However, it was not reported among the protein components of purified Tetrahymena telomerase, the first telomerase identified and the most thoroughly studied. It therefore seemed possible that Tetrahymena used an alternative telomerase that lacked a TERT protein. We now report the cloning and sequencing of a Tetrahymena thermophila gene whose encoded protein has the properties expected for a TERT, including large size (133 kDa), basicity (calculated pI = 10.0), and reverse transcriptase sequence motifs with telomerase-specific features. The expression of mRNA from the Tetrahymena TERT gene increases dramatically at 2-5 h after conjugation, preceding de novo addition of telomeres to macronuclear DNA molecules. We also report the cloning and sequencing of the ortholog from Oxytricha trifallax. The Oxytricha macronuclear TERT gene has no introns, whereas that of Tetrahymena has 18 introns. Sequence comparisons reveal a new amino acid sequence motif (CP), conserved among the ciliated protozoan TERTs, and allow refinement of previously identified motifs. A phylogenetic tree of the known TERTs follows the phylogeny of the organisms in which they are found, consistent with an ancient origin rather than recent transposition. The conservation of TERTs among eukaryotes supports the model that telomerase has a conserved core (TERT plus the RNA subunit), with other subunits of the holoenzyme being more variable among species.

Telomere length dynamics in telomerase-positive immortal human cell populations.

It has been proposed that the progressive shortening of telomeres in somatic cells eventually results in senescence. Previous experiments have demonstrated that many immortal cell lines have acquired telomerase activity leading to stabilization of telomere length. Telomere dynamics and telomerase activity were examined in the telomerase-positive immortal cell lines HeLa and 293 and subclones derived from them. A mass culture of HeLa cells had a stable mean telomere length over 60 population doublings (PD) in vitro. Subclones of this culture, however, had a range of mean telomere lengths indicating that telomeric heterogeneity exists within a population with a stable mean telomere length. Some of the subclones lacked detectable telomerase activity soon after isolation but regained it by PD 18, suggesting that at least some of the variation in telomere length can be attributed to variations in telomerase activity levels. 293 subclones also varied in telomere length and telomerase activity. Some telomerase-positive 293 subclones contained long telomeres that gradually shortened, demonstrating that factors other than telomerase also act to modulate telomere length. Fluctuations in telomere length in telomerase-positive immortalized cells may contribute to chromosomal instability and clonal evolution.

Evidence for an alternative mechanism for maintaining telomere length in human tumors and tumor-derived cell lines.

The gradual loss of DNA from the ends of telomeres has been implicated in the control of cellular proliferative potential. Telomerase is an enzyme that restores telomeric DNA sequences, and expression of its activity was thought to be essential for the immortalization of human cells, both in vitro and in tumor progression in vivo. Telomerase activity has been detected in 50-100% of tumors of different types, but not in most normal adult somatic tissues. It has also been detected in about 70% of human cell lines immortalized in vitro and in all tumor-derived cell lines examined to date. It has previously been shown that in vitro immortalized telomerase-negative cell lines acquire very long and heterogeneous telomeres in association with immortalization presumably via one or more novel telomere-lengthening mechanisms that we refer to as ALT (alternative lengthening of telomeres). Here we report evidence for the presence of ALT in a subset of tumor-derived cell lines and tumors. The maintenance of telomeres by a mechanism other than telomerase, even in a minority of cancers, has major implications for therapeutic uses of telomerase inhibitors.

Normal telomere maintenance in immortal ataxia telangiectasia cell lines.

Telomeres are maintained in germ line cells and immortal cell lines, but shorten with each cell division in most somatic cells. Blood lymphocytes from individuals with ataxia telangiectasia (AT) demonstrate an accelerated rate of telomere shortening and high levels of telomere associations. This accelerated loss of telomeres in somatic cells in AT could be due to either the loss of more telomeric DNA with every cell division or an increased rate of cell division. The gene for AT shares homology with the yeast TEL1 gene, in which mutations result in abnormally shortened telomeres. Thus, mutations in the gene for ataxia telangiectasia may also influence the ability of germ line cells and immortal cell lines to properly maintain telomere homeostasis. To investigate a possible defect of telomere maintenance in AT we have analyzed 8 simian virus 40 (SV40)-immortalized AT cell lines and twelve SV40-immortalized non-AT cell lines for both telomerase activity and telomere length. The results demonstrate that telomere length in AT cells is maintained via telomerase or an alternative (ALT) pathway in a manner indistinguishable from cell lines derived from normal cells. We also investigated telomere dynamics in one telomerase-positive AT cell line by analyzing the changes in the length of a single telomere, and found that this telomere maintained its equilibrium mean length (EML) similar to normal cell lines with stable chromosomes. The combined results show no significant differences between the telomeres of immortal AT and non-AT cell lines, demonstrating that the absence of wild-type ATM does not result in a fundamental defect in telomere maintenance in these cells.

The telomere lengthening mechanism in telomerase-negative immortal human cells does not involve the telomerase RNA subunit.

According to the telomere hypothesis of senescence, the progressive shortening of telomeres that occurs upon division of normal somatic cells eventually leads to cellular senescence. The immortalisation of human cells is associated with the acquisition of a telomere maintenance mechanism which is usually dependent upon expression of the enzyme telomerase. About one third of in vitro immortalised human cell lines, however, have no detectable telomerase but contain telomeres that are abnormally long. The nature of the alternative telomere maintenance mechanism (referred to as ALT, for Alternative Lengthening of Telomeres) that must exist in these telomerase-negative cells has not been elucidated. It has previously been shown that abnormal lengthening of yeast telomeres may occur due to mutations in the yeast telomerase RNA gene. That this is not the mechanism of the abnormally long telomeres in ALT cell lines was demonstrated by the finding that seven of seven ALT lines have wild-type human telomerase RNA (hTR) sequence, including a novel polymorphism that is present in 30% of normal individuals. We found that two ALT cell lines have no detectable expression of the hTR gene. This shows that the ALT mechanism in these cell lines is not dependent on hTR. Expression of exogenous hTR via infection of these cells with a recombinant hTR-adenovirus vector did not result in telomerase activity, indicating that their lack of telomerase activity is not due to absence of hTR expression. We conclude that the ALT mechanism is not dependent on the expression of hTR, and does not involve mutations in the hTR sequence.

Telomere dynamics and telomerase activity in in vitro immortalised human cells.

This article reviews the current understanding of the involvement of telomerase in in vitro immortalisation of human cells. In vitro immortalisation with DNA tumour viruses or chemicals usually occurs in two phases. The first stage is an extension of lifespan beyond that at which cells would normally senescence, after which the culture enters a period of crisis. The second stage involves the escape from crisis of a rare cell in the culture, which goes on to proliferate indefinitely. The hypothesis that telomere shortening acts as a signal for senescence and crisis, and that cells need to activate telomerase to survive these states, gained support from early studies examining telomere behaviour and telomerase activity in immortalised cell lines. In many cases, telomeres were found to continue to shorten during the phase of extended lifespan, and no telomerase was detectable. Cells which survived crisis had activated telomerase and had stable or lengthened telomerase. However, it is now clear that this model does not apply to all cell lines. Approximately a quarter of in vitro immortalised cell lines so far examined have no detectable telomerase activity, yet have very long and heterogeneous telomeres. These cell lines have acquired a novel mechanism for lengthening their telomeres, named ALT (Alternative Lengthening of Telomeres). The nature of ALT is not yet understood, but may involve non-reciprocal recombination between telomeres. ALT is not merely a phenomenon of in vitro immortalised cell lines, but has also been found in tumours and tumour-derived cell lines. Furthermore, there are a number of cell lines which have been shown to have low levels of telomerase prior to crisis while telomere shortening is still occurring, and the function of these low levels of telomerase activity is unknown.

Immortalized cells with no detectable telomerase activity. A review.

Immortalization of human cells in culture is usually associated with expression of telomerase activity. In some cases, however, no telomerase activity is detectable even though comparison of the terminal restriction fragment (TRF) pattern before and after immortalization shows that lengthening of telomeres has occurred. The extreme heterogeneity in telomere length and the differences in the dynamics of telomere maintenance in telomerase-negative cell lines compared to telomerase-positive cell lines indicate that these cells have utilized one or more alternative mechanisms for lengthening of telomeres (ALT). All telomerase-negative immortalized cell lines examined to date show evidence of ALT activity, consistent with the hypothesis that telomere maintenance either by telomerase or by ALT is required for immortalization. The nature of the ALT mechanism(s) is currently unknown, but studies of telomere dynamics in an ALT cell line containing a marker just proximal to the telomeric sequences show gradual shortening of the telomere followed by rapid elongation. This is consistent with a non-reciprocal recombinational mechanism similar to that found in telomerase-defective mutant yeast strains.

Association of extended in vitro proliferative potential with loss of p16INK4 expression.

This study addresses the question of whether loss of p16INK4 expression contributes to the immortalization of human cells. In vitro immortalization usually proceeds through two phases. In the first phase (lifespan extension), cells continue proliferating and their telomeres continue shortening beyond the point at which normal cells become senescent. In the second phase (immortalization), the cells activate a telomere maintenance mechanism and acquire an unlimited proliferative potential. It has previously been shown that immortalized cells containing viral oncoproteins that bind and inactivate p110RB contain wild-type p16INK4; we therefore examined the p16INK4 status of cell lines that became immortalized in vitro in the absence of these oncoproteins. Three such lines were identified: III-CF/.2A1 and III-CF/E6A2 (both derived from Li-Fraumeni syndrome fibroblasts, probably by spontaneous immortalization) and MePV-231 (normal mesothelial cells transfected with HPV-16 E6/E7 genes that underwent deletion of these genes before immortalization). In each case p16INK4 expression was lost at or before immortalization. Further, a cell strain was identified that had an extended but finite lifespan associated with loss of p16INK4 (and p53) expression. Thus loss of p16INK4 expression was associated with extended in vitro lifespan but was not sufficient for immortalization, even in the absence of wild-type p53.

Involvement of RB-1, p53, p16INK4 and telomerase in immortalisation of human cells.

Involvement of the retinoblastoma susceptibility (RB-1), p16INK4, p53 and telomerase genes in immortalisation was examined by determining their status in 15 human cell lines representing four immortalisation complementation groups. No abnormalities of RB-1, p53 and p16INK4 were detected in cell lines containing DNA tumour virus proteins known to bind to the protein products of the RB-1 and p53 genes. In contrast, in all other cell lines from each of the four groups either RB-1 was mutant or p16INK4 protein was undetectable and there were cell lines containing p53 mutations in three of the groups. Telomerase activity was detected in 12/15 lines, including some of the virally immortalised lines and in some lines from each group. Since none of these changes correlated with complementation group, other genetic changes must be required for immortalisation.

Alterations in p53 and p16INK4 expression and telomere length during spontaneous immortalization of Li-Fraumeni syndrome fibroblasts.

Normal cells have a strictly limited growth potential and senesce after a defined number of population doublings (PDs). In contrast, tumor cells often exhibit an apparently unlimited proliferative potential and are termed immortalized. Although spontaneous immortalization of normal human cells in vitro is an extremely rare event, we observed this in fibroblasts from an affected member of a Li-Fraumeni syndrome kindred. The fibroblasts were heterozygous for a p53 mutation and underwent senescence as expected at PD 40. In four separate senescent cultures (A to D), there were cells that eventually recommenced proliferation. This was associated with aneuploidy in all four cultures and either loss (cultures A, C, and D) or mutation (culture B) of the wild-type (wt) p53 allele. Loss of wt p53 function was insufficient for immortalization, since cultures A, B, and D subsequently entered crisis from which they did not escape. Culture C has continued proliferating beyond 400 PDs and thus appears to be immortalized. In contrast to the other cultures, the immortalized cells have no detectable p16INK4 protein. A culture that had a limited extension of proliferative potential exhibited a progressive decrease in telomere length with increasing PD. In the culture that subsequently became immortalized, the same trend occurred until PD 73, after which there was a significant increase in the amount of telomeric DNA, despite the absence of telomerase activity. Immortalization of these cells thus appears to be associated with loss of wt p53 and p16INK4 expression and a novel mechanism for the elongation of telomeres.

Telomere elongation in immortal human cells without detectable telomerase activity.

Immortalization of human cells is often associated with reactivation of telomerase, a ribonucleoprotein enzyme that adds TTAGGG repeats onto telomeres and compensates for their shortening. We examined whether telomerase activation is necessary for immortalization. All normal human fibroblasts tested were negative for telomerase activity. Thirteen out of 13 DNA tumor virus-transformed cell cultures were also negative in the pre-crisis (i.e. non-immortalized) stage. Of 35 immortalized cell lines, 20 had telomerase activity as expected, but 15 had no detectable telomerase. The 15 telomerase-negative immortalized cell lines all had very long and heterogeneous telomeres of up to 50 kb. Hybrids between telomerase-negative and telomerase-positive cells senesced. Two senescent hybrids demonstrated telomerase activity, indicating that activation of telomerase is not sufficient for immortalization. Some hybrid clones subsequently recommenced proliferation and became immortalized either with or without telomerase activity. Those without telomerase activity also had very long and heterogeneous telomeres. Taken together, these data suggest that the presence of lengthened or stabilized telomeres is necessary for immortalization, and that this may be achieved either by the reactivation of telomerase or by a novel and as yet unidentified mechanism.

SV40-induced immortalization of human cells.

For several decades simian virus 40 (SV40) early region genes have been used as a means of generating immortalized human cell lines; however, the molecular mechanisms of this process have begun to be understood only recently. SV40-induced immortalization proceeds via two phases. In the first phase ("lifespan extension"), cells continue proliferating for a limited number of population doublings beyond the point at which normal cells undergo senescence. This is mainly due to the ability of SV40 large T antigen (LTAg) to bind to the protein products of the p53 and retinoblastoma (Rb) genes. The second phase ("immortalization") occurs in only a small minority of cells, and cell hybridization analyses indicate that this is a gene inactivation event. The gene or genes involved are currently unknown, but chromosomal localization data are accumulating which should make their cloning and characterization possible in the near future.