Polymorphic tandem DNA repeats activate the human telomerase reverse transcriptase gene.

Multiple independent sequence variants of the hTERT locus have been associated with telomere length and cancer risks in genome-wide association studies. Here, we identified an intronic variable number tandem repeat, VNTR2-1, as an enhancer-like element, which activated hTERT transcription in a cell in a chromatin-dependent manner. VNTR2-1, consisting of 42-bp repeats with an array of enhancer boxes, cooperated with the proximal promoter in the regulation of hTERT transcription by basic helix-loop-helix transcription factors and maintained hTERT expression during embryonic stem-cell differentiation. Genomic deletion of VNTR2-1 in MelJuSo melanoma cells markedly reduced hTERT transcription, leading to telomere shortening, cellular senescence, and impairment of xenograft tumor growth. Interestingly, VNTR2-1 lengths varied widely in human populations; hTERT alleles with shorter VNTR2-1 were underrepresented in African American centenarians, indicating its role in human aging. Therefore, this polymorphic element is likely a missing link in the telomerase regulatory network and a molecular basis for genetic diversities of telomere homeostasis and age-related disease susceptibilities.

Phase-Separated Dielectric Gels Based on Christiansen Effect.

Phase separation is a trivial phenomenon but a mature strategy in materials science. The flexible materials are provided toughness and strength by phase separation, yet there are few applications in optics and electronics industry. A novel phase-separated dielectric gel (PSDG) with a strong Christiansen effect is prepared via radical polymerization using hydroxyethyl methacrylate as a monomer, 4-cyano-4'-pentylbiphenyl and tributyl citrate as mixed solvents, and polyethylene glycol as a softener. The solvent ratios and ambient conditions can efficiently change the color of PSDG which makes it strongly selective for the wavelength of transmitted light. Besides, it has a high dielectric constant (10 at 1 kHz), sensitively responding to the electric field. The phase separation degree of PSDG varies with applied electric field, which will induce its transmittance alteration accordingly. The current field sensitive PSDG provides a novel idea for "smart windows". Additionally, varying the size and shape of the electrodes can precisely control the phase separation in PSDG and also enables the function of free writing on flexible materials. Therefore, the designed PSDG has great application potential for flexible touch and interesting interactions.

Morphological and genetic analysis of Ceratomyxa saurida Zhao et al. 2015 and Ceratomyxa mai sp. nov. (Myxozoa: Ceratomyxidae) from the East China Sea.

We describe Ceratomyxa saurida Zhao et al. 2015 and Ceratomyxa mai sp. nov. (Myxozoa: Ceratomyxidae) from the East China Sea. C. saurida was found in the gallbladders of 3/13 specimens of its type host, Saurida elongata Temminck and Schlegel 1846 (Aulopiformes). Myxospore characters were consistent with the original description to which we have added small subunit (SSU) rRNA gene data. C. mai sp. nov. was found in gallbladders of 3/13 specimens of S. elongata and 5/13 specimens of Neobythites sivicola Jordan and Snyder 1901 (Ophidiiformes). Mature myxospores of C. mai sp. nov. were crescentic in sutural view, with a deeply concave posterior angle 142.2±8.2° (125.8‒158.2°) and an arched anterior side. Shell valves were smooth and equal, 20.9±1.9 (17.3‒24.7) µm thick and 9.2±0.5 (8.1‒9.9) µm long, and joined at a straight, thin sutural plane passing between two nematocysts (polar capsules). The nematocysts were equal-sized, pyriform, 2.6±0.2 (2.4‒2.9) µm long and 2.7±0.2 (2.4‒3.3) µm wide, with their tapered ends pointed toward each other, located in the anterior third of the spore. Sequences of the SSU rRNA gene and internal transcribed spacer 1 showed that the isolates of C. mai sp. nov. obtained from S. elongata and N. sivicola were identical. The SSU rRNA gene sequence of C. mai sp. nov. was distinct from all known myxosporeans and clustered with C. saurida, and then with Ceratomyxa filamentosi Kalatzis, Kokkari and Katharios 2013, both of which also infect Aulopiformes fishes.

Myxobolus xiushanensis n. sp. (Myxozoa: Myxobolidae) infecting the gill arches of Yellowhead Catfish Tachysurus fulvidraco from China.

OBJECTIVE: Myxosporidiosis of bagrid fishes has been a focus of aquaculture research in recent years. The purpose of this study is to characterize a novel myxobolid, named Myxobolus xiushanensis n. sp., infecting Yellowhead Catfish Tachysurus fulvidraco in China. METHODS: We used molecular biology, morphology, phylogeny, and histopathology in the present study. RESULT: Mature myxospores were circular to ellipsoidal in valve view, measuring 12.2 ± 0.4 µm (mean ± SD; range = 11.2-13.2 µm) in length and 10.6 ± 0.4 µm (9.5-11.1 µm) in width. Two oval polar capsules were equal in width (3.4 ± 0.2 µm; 3.0-3.8 µm) but slightly unequal in length: 5.6 ± 0.3 µm (5.3-6.1 µm) and 4.7 ± 0.2 µm (4.4-5.5 µm). The polar capsule was packed with five to seven spirals of polar tubules. Histopathological investigation demonstrated that the plasmodium under the cuticular layer of the gill arch only induced a local inflammatory response and did not cause serious damage to the gill arch's internal structure. The two small subunit (SSU) ribosomal DNA sequences of M. xiushanensis n. sp. showed 100% similarity and uniqueness, and the highest similarity with other myxosporean sequences in GenBank was 90.27% (query coverage = 94%). The secondary structures of the SSU ribosomal RNA revealed that the present species was distinctly different from related species in regions V4 and V7. Phylogenetic analysis showed that M. xiushanensis n. sp. clustered independently within a branch. CONCLUSION: These results enrich our understanding of the biodiversity of myxobolids infecting bagrid fishes and provide fundamental data for the diagnosis of myxosporidiosis.

Multiple Dynamic Bonds-Driven Integrated Cathode/Polymer Electrolyte for Stable All-Solid-State Lithium Metal Batteries.

All-solid-state lithium metal batteries (LMBs) are considered as the promising higher-energy and improved-safety energy-storage systems. Nevertheless, the electrolyte-electrodes interfacial issues due to the limited solid physical contact lead to discontinuous interfacial charge transport and large interfacial resistance, thereby suffering from unsatisfactory electrochemical performance. Herein, we construct an integrated cathode/polymer electrolyte for all-solid-state LMBs under the action of polymer chains exchange and recombination originating from multiple dynamic bonds in our well-designed dynamic supramolecular ionic conductive elastomers (DSICE) molecular structure. The DSICE acts as polymer electrolytes with excellent electrochemical performance and mechanical properties, achieving the ultrathin pure polymer electrolyte thickness (12 µm). Notably, the DSICE also functions as lithium iron phosphate (LiFePO4 , LFP) cathode binders with enhanced adhesive capability. Such well-constructed Li|DSICE|LFP-DSICE cells generate delicate electrolyte-electrodes interfacial contact at the molecular level, providing continuous Li+ transport pathways and promoting uniform Li+ deposition, further delivering superior long-term charge/discharge stability (>600 cycles, Coulombic efficiency, >99.8 %) and high capacity retention (80 % after 400 cycles). More practically, the Li|DSICE|LFP-DSICE pouch cells show stable electrochemical performance, excellent flexibility and safety under abusive tests.

Human Cytomegalovirus Utilizes Extracellular Vesicles To Enhance Virus Spread.

Human cytomegalovirus (HCMV) manipulates cellular processes associated with secretory pathways within an infected cell to facilitate efficient viral replication. However, little is known about how HCMV infection alters the surrounding cellular environment to promote virus spread to uninfected cells. Extracellular vesicles (EVs) are key signaling molecules that are commonly altered in numerous disease states. Previous reports have shown that viruses commonly alter EVs, which can significantly impact infection. This study finds that HCMV modulates EV biogenesis machinery through upregulation of the endosomal sorting complex required for transport (ESCRT) proteins. This regulation appears to increase the activity of EV biogenesis, since HCMV-infected fibroblasts have increased vesicle release and altered vesicle size compared to EVs from uninfected cells. EVs generated through ESCRT-independent pathways are also beneficial to virus spread in fibroblasts, as treatment with the EV inhibitor GW4869 slowed the efficiency of HCMV spread. Importantly, the transfer of EVs purified from HCMV-infected cells enhanced virus spread. This suggests that HCMV modulates the EV pathway to transfer proviral signals to uninfected cells that prime the cellular environment for incoming infection and enhance the efficiency of virus spread.IMPORTANCE Human cytomegalovirus (HCMV) is a herpesvirus that leads to serious health consequences in neonatal or immunocompromised patients. Clinical management of infection in these at-risk groups remains a serious concern even with approved antiviral therapies available. It is necessary to increase our understanding of the cellular changes that occur during infection and their importance to virus spread. This may help to identify new targets during infection that will lead to the development of novel treatment strategies. Extracellular vesicles (EVs) represent an important method of intercellular communication in the human host. This study finds that HCMV manipulates this pathway to increase the efficiency of virus spread to uninfected cells. This finding defines a new layer of host manipulation induced by HCMV infection that leads to enhanced virus spread.

A validated analysis pipeline for mass spectrometry-based vitreous proteomics: new insights into proliferative diabetic retinopathy.

BACKGROUND: Vitreous is an accessible, information-rich biofluid that has recently been studied as a source of retinal disease-related proteins and pathways. However, the number of samples required to confidently identify perturbed pathways remains unknown. In order to confidently identify these pathways, power analysis must be performed to determine the number of samples required, and sample preparation and analysis must be rigorously defined. METHODS: Control (n = 27) and proliferative diabetic retinopathy (n = 23) vitreous samples were treated as biologically distinct individuals or pooled together and aliquoted into technical replicates. Quantitative mass spectrometry with tandem mass tag labeling was used to identify proteins in individual or pooled control samples to determine technical and biological variability. To determine effect size and perform power analysis, control and proliferative diabetic retinopathy samples were analyzed across four 10-plexes. Pooled samples were used to normalize the data across plexes and generate a single data matrix for downstream analysis. RESULTS: The total number of unique proteins identified was 1152 in experiment 1, 989 of which were measured in all samples. In experiment 2, 1191 proteins were identified, 727 of which were measured across all samples in all plexes. Data are available via ProteomeXchange with identifier PXD025986. Spearman correlations of protein abundance estimations revealed minimal technical (0.99-1.00) and biological (0.94-0.98) variability. Each plex contained two unique pooled samples: one for normalizing across each 10-plex, and one to internally validate the normalization algorithm. Spearman correlation of the validation pool following normalization was 0.86-0.90. Principal component analysis revealed stratification of samples by disease and not by plex. Subsequent differential expression and pathway analyses demonstrated significant activation of metabolic pathways and inhibition of neuroprotective pathways in proliferative diabetic retinopathy samples relative to controls. CONCLUSIONS: This study demonstrates a feasible, rigorous, and scalable method that can be applied to future proteomic studies of vitreous and identifies previously unrecognized metabolic pathways that advance understanding of diabetic retinopathy.

Descriptive analysis of Fibulin-3 and the extracellular vesicle marker, Alix, in drusen from a small cohort of postmortem human eyes.

Fibulin-3 (Fib3) is a secreted glycoprotein that is expressed in the retina and has been associated with drusen formation in age-related macular degeneration (AMD). The purpose of this study was to assess whether Fib3 is associated with extracellular vesicles (EVs) in drusen from non-diseased and AMD human donors. De-identified sections of human eyes were received from the National Disease Research Institute (NDRI, Philadelphia). Donor eyes were either non-diseased (no known ocular pathology) or had been diagnosed with AMD. Retinal cryostat sections were labeled with primary antibodies targeted to Fib3, Apolipoprotein E (ApoE; a drusen marker), and ALG-2 interacting protein X (Alix, an EV marker) for confocal imaging (Leica TCS SP8). Fib3-positive (Fib3+) puncta were detected on the apical region of the RPE layer and within large AMD drusen. Alix-positive (Alix+) puncta were also detected in a single AMD druse, where a number were Fib3+ and the remaining were Fib3-negative. Similarly, there were Fib3+ puncta that were Alix-negative. Fib3 and Alix also showed a degree of colocalization in the photoreceptor outer segments of the neural retina. Our data suggest that the Alix+ puncta are EV-rich populations that accumulate, together with Fib3, within the drusen matrix during AMD. The EV population is likely heterogeneous, such that there are sub-populations with different cargo content.

Description of Myxidium pseudocuneiforme n. sp. (Myxosporea: Myxidiidae) from Cyprinus carpio in China, with the resolution on a taxonomic dilemma of Myxidium cuneiforme.

Investigations on myxozoan parasites of fish from Chongqing in China, revealed two Myxidium cuneiforme-like myxosporeans infecting the gallbladder of Cyprinus carpio carpio and Carassius auratus. We researched their myxospore morphology, and analyzed their genetic similarity and phylogenic relationships to other myxozoans based on small subunit ribosomal DNA (18S rDNA) sequences. Although both parasites recovered were morphologically similar, the myxosporean isolated from C. auratus was consistent in morphology to Myxidium cuneiforme, which was described from this host species. The parasite isolated from C. c. carpio had overlapping myxospore dimensions to M. cuneiforme, but on average, the polar capsules were not as long. More importantly, this parasite was genetically distinct from M. cuneiforme with 96.3% and 96.5% similarity in two sequences of 18S rDNA, and we propose the name Myxidium pseudocuneiforme n. sp. for this myxozoan from common carp. Its mature myxospores are ellipsoidal and asymmetric with pointed ends in valvular view, arc-shaped or fusiform in sutural view. The pyriform polar capsules are equal in size, and polar filament with 5-6 coils. This study highlights that molecular characteristics and host specificity are indispensable for myxozoan species identification when presented with the taxonomic dilemma of whether we are observing one species that exhibits slight morphological differences or multiple, but similar, species in different hosts.

Molecular characterization and phylogenetic analyses of Tripartiella macrosoma Basson and Van As, 1987 and Tripartiella obtusa Ergens and Lom, 1970 based on 18S rRNA gene sequence data.

In the present study, we provided the first 18S rRNA gene sequence data of two Tripartiella species, Tripartiella macrosoma Basson and Van As, 1987 and Tripartiella obtusa Ergens and Lom, 1970, which were isolated from Tachysurus fulvidraco (Richardson, 1846) and Hemibarbus maculatus Bleeker, 1871 in Chongqing, China, respectively. Morphologically, both species fall within the morphometry range of the original descriptions and are very similar to the original populations in the overall appearance of the adhesive disc. Tripartiella macrosoma can be easily distinguished from the other Tripartiella species by possessing the denticle with a long strip and conspicuously inclined backward blade and a robust and short ray. Tripartiella obtusa is mainly characterized by a broad blade and a relatively long ray. Phylogenetically, T. macrosoma clustered with Trichodinella myakkae (Mueller, 1937) Raabe, 1950 and further with Trichodinella sp., which was sister to a group that includes four populations of Trichodinella epizootica (Raabe, 1950) Srámek-Husek, 1953; finally, they formed a small clade with T. obtusa. This result suggested that T. macrosoma had a closer relationship with Trichodinella spp. than with T. obtusa and T. obtusa diverged earlier than T. macrosoma and Trichodinella spp. By combining morphological and molecular data, the polyphyletic characteristics of Tripartiella and Trichodinella were further analyzed, and the results revealed that the validity of the genus Tripartiella is doubtful.

Characterization of Myxidium spinibarba sp. nov. (Cnidaria, Myxosporea, Myxidiidae) from Spinibarbus sinensis (Bleeker, 1871) in Chongqing China.

In the present study, we described a new species of Myxidium Bütschli, 1882, obtained from the gallbladder of Spinibarbus sinensis (Bleeker, 1871) from the Jialing River in Chongqing, China. Myxidium spinibarba sp. nov. was identified based on morphological and SSU rDNA sequence data. The mature myxospores were fusiform in valvular view and ovoid in sutural view, with somewhat protrusive poles and mean dimensions (all in µm) of 11.8 ± 0.5 (10.6-12.4) in length and 6.1 ± 0.5 (5.5-7.2) in width. The polar capsules were pyriform and equal in size with mean dimensions of 3.6 ± 0.4 (3.0-4.4) in length and 3.0 ± 0.2 (2.7-3.2) in width. The new species was distinct from related species of Myxidium in its morphology and molecular characteristics. Phylogenetic analysis indicated the clustering of species based on the presence or absence of valvular striations. Moreover, myxospore morphology, rather than the host environment, played an important role in the partial phylogenetic clustering.

Taxonomy on three novel species of Sphaeromyxa Thélohan 1892 (Myxozoa, Bivalvulida, Sphaeromyxidae) with insight into the evolution of the genus.

Three new myxosporeans of the genus Sphaeromyxa Thélohan 1892 were discovered from the coastal waters off Xiamen in the East China Sea and characterized based on morphological and SSU rDNA data. Sphaeromyxa photopectoralis sp. n. was described from Photopectoralis bindus, and Sphaeromyxa sebastisca sp. n. was described infecting both Sebastiscus marmoratus (type-host) and Scorpaenopsis cirrosa. These two species are morphologically consistent with the "balbianii" group, possessing straight myxospores and truncated ends, but are distinct from one another genetically and by myxospore dimensions. A third myxosporean infecting Siganus fuscescens was described as Sphaeromyxa xiamenensis sp. n., and this species is morphologically consistent with the "incurvata" group, bearing arcuate myxospores with rounded ends. The molecular phylogeny and estimated rRNA secondary structure suggest that marine sphaeromyxids are probably derived from freshwater myxidiids, and "incurvata" and "balbianii" groups might each represent independent evolutionary lineages. The present study also shows that S. limocapitis phylogenetically nested in "incurvata" group.

The first report of two Sphaeromyxa species (Myxozoa: Bivalvulida) from the South China Sea: Sphaeromyxa scorpaena n. sp. from long-fingered scorpionfish (Scorpaenodes albaiensis) and Sphaeromyxa theraponi from tiger perch (Terapon jarbua).

Two myxosporean species of the genus Sphaeromyxa were isolated from the gallbladders of marine fish in the South China Sea. Sphaeromyxa scorpaena n. sp. was collected from Scorpaenodes albaiensis Evermann and Seale, 1907. The mature myxospores were arcuate-shaped with tapered to pointed ends, and a length of 14.1 ± 0.7 (13.8-15.1) µm and a width of 5.2 ± 0.3 (4.9-5.8) µm. The polar capsules (PCs) were pyriform with a length of 3.2 ± 0.2 (3.1-3.5) µm and a width of 1.6 ± 0.1 (1.4-1.8) µm, and containing ribbon-like polar filaments irregularly folded 1.5-2.5 turns. Molecular characteristics and phylogenetic analysis based on 18S rDNA as well as morphological comparison confirmed that S. scorpaena n. sp. was a previously undescribed species. Sphaeromyxa theraponi, isolated from Terapon jarbua Forsskål, 1775, was reported for the first time from the South China Sea. The mature myxospores were slightly arched, tapering to bluntly rounded ends, with a length of 17.3 ± 0.9 (15.5-19.4) µm and a width of 4.8 ± 0.3 (4.1-5.3) µm. A sporoplasm was situated in the space between PCs in the myxospore. The PCs were pyriform, which contained ribbon-like polar filaments irregularly folded by 2-3 turns, with a length of 7.0 ± 0.5 (5.8-8.1) µm and a width of 2.6 ± 0.2 (2.2-3.0) µm. Our morphological and phylogenetic analyses suggest that the pointed ends of S. scorpaena n. sp. might be a secondarily acquired characteristic rather than an ancestral trait.

Morphological Redescription and Molecular Identification of Trichodina reticulata Hirschmann & Partsch, 1955 (Ciliophora, Mobilida, Trichodinidae) with the Supplemental New Data of SSU rDNA and ITS-5.8S rDNA.

During a survey of trichodinids in Chongqing, China, two populations of Trichodina reticulata Hirschmann & Partsch, 1955 were isolated from gills of the freshwater fishes, Carassius auratus and Ctenopharyngodon idellus, and 11 molecular samples containing small subunit (SSU) rDNA and internal transcribed spacer (ITS)-5.8S rDNA were newly sequenced. Both populations fell within the range of morphometry and corresponded well with the diagnostic morphological features of Trichodina reticulata Hirschmann & Partsch, 1955. It should be noted that one population possessed obvious central granules in the adhesive disc, while the other one did not. The detailed morphological redescription for these two populations is given in this work. Although some intraspecific differences were found, both populations were confirmed as Trichodina reticulata based on the morphological study and molecular data, including the sequence analysis of the primary and secondary structures of SSU rDNA, genetic distances and phylogenetic tree. Our study supports the assertion that central granules in the adhesive disc might be an auxiliary feature rather than a heritable character for determining taxonomic affiliations among trichodinids.

The impacts of geographic and host species isolation on population divergence of Myxobolus lentisuturalis.

Samples of Myxobolus lentisuturalis were found in the gallbladder of Carassius auratus in Chongqing, China, without obvious disease symptoms, which were different from samples reported in Hubei, China, and Italy which were described as highly pathogenic muscle-infecting species. In order to improve our understanding of the relationships between these different samples, we analyzed geography, DNA sequence data, and site specificity. The results indicated that (1) the genetic relationship between Chongqing and Italy samples of M. lentisuturalis was much closer than relationship between each of them and the Hubei samples; (2) host species isolation was more important than the geographic isolation in divergence of M. lentisuturalis samples, and the species might be specialized among its different host species; and (3) geographic isolation and infection-site variation played a limited impact in genetic differentiation among different samples of M. lentisuturalis infecting the same host species.

Repression of telomerase gene promoter requires human-specific genomic context and is mediated by multiple HDAC1-containing corepressor complexes.

The human telomerase reverse transcriptase (hTERT) gene is repressed in most somatic cells, whereas the expression of the mouse mTert gene is widely detected. To understand the mechanisms of this human-specific repression, we constructed bacterial artificial chromosome (BAC) reporters using human and mouse genomic DNAs encompassing the TERT genes and neighboring loci. Upon chromosomal integration, the hTERT, but not the mTert, reporter was stringently repressed in telomerase-negative human cells in a histone deacetylase (HDAC)-dependent manner, replicating the expression of their respective endogenous genes. In chimeric BACs, the mTert promoter became strongly repressed in the human genomic context, but the hTERT promoter was highly active in the mouse genomic context. Furthermore, an unrelated herpes simplex virus-thymidine kinase (HSV-TK) promoter was strongly repressed in the human, but not in the mouse, genomic context. These results demonstrated that the repression of hTERT gene was dictated by distal elements and its chromatin environment. This repression depended on class I HDACs and involved multiple corepressor complexes, including HDAC1/2-containing Sin3B, nucleosome remodeling and histone deacetylase (NuRD), and corepressor of RE1 silencing transcription factor (CoREST) complexes. Together, our data indicate that the lack of telomerase expression in most human somatic cells results from its repressive genomic environment, providing new insight into the mechanism of long-recognized differential telomerase regulation in mammalian species.-Cheng, D., Zhao, Y., Wang, S., Zhang, F., Russo, M., McMahon, S. B., Zhu, J. Repression of telomerase gene promoter requires human-specific genomic context and is mediated by multiple HDAC1-containing corepressor complexes.

A New Molecular Approach Based on the Secondary Structure of Ribosomal RNA for Phylogenetic Analysis of Mobilid Ciliates.

We analyzed the secondary structure of the small subunit (SSU) rRNA genes of Mobilida (Ciliophora, Peritrichia) and found that the secondary structures of some regions within the SSU-rRNA gene are distinct between the families Trichodinidae and Urceolariidae. Therefore, some of these important regions including H10, H11, H17, H47, H29, H30, H37, E10-1, H45-H46, and V4 (E23-4, E23-7) could be used as the barcodes for classification of these two families. In contrast, V4 (E23-1, E23-2) belongs to a hypervariable region and is not a good barcode at the genus level because of its great inter-specific variation. Our results indicated that the comprehensive analysis of the secondary structure of SSU-rRNA genes is a reliable auxiliary approach for phylogenic study of mobilid ciliates. It was further found that the coevolution between hosts or habitats and the Mobilida ciliates was existent, because the host types and their habitats were critical ecological factors that influenced the evolution of Mobilida ciliates.

Morphological and Molecular Identification of the New Species, Trichodina pseudoheterodentata sp. n. (Ciliophora, Mobilida, Trichodinidae) from the Channel Catfish, Ictalurus punctatus, in Chongqing China.

It is difficult to differentiate similar trichodinids solely based on morphological examination, thus other identification methods, such as molecular identification, are necessary for identification. One mobilid ciliate named Trichodina pseudoheterodentata sp. n. was isolated from the gills of channel catfish, Ictalurus punctatus, in Chongqing, China. In the present study, its SSU rDNA was sequenced for the first time. Based on the results from both morphological identification and SSU rDNA sequencing, the new species was identified and compared with similar species. The morphological analysis revealed that T. pseudoheterodentata is a large Trichodina species (cell diameter 73.0-82.5 µm) and possesses robust denticles with broad blades and well-developed blade connections. Characterization of its primary and secondary SSU rDNA structures indicated that T. pseudoheterodentata was distinctly different from congeneric species in H12, H15, E10_1, and V4 regions. Phylogenetic analysis revealed that the genetic distances among the new species and similar species reached interspecific levels, furthermore, the phylogenetic study also validated the identification of T. pseudoheterodentata and its placement in the genus Trichodina.

Human Telomerase Reverse Transcriptase (hTERT) Transcription Requires Sp1/Sp3 Binding to the Promoter and a Permissive Chromatin Environment.

The transcription of human telomerase gene hTERT is regulated by transcription factors (TFs), including Sp1 family proteins, and its chromatin environment. To understand its regulation in a relevant chromatin context, we employed bacterial artificial chromosome reporters containing 160 kb of human genomic sequence containing the hTERT gene. Upon chromosomal integration, the bacterial artificial chromosomes recapitulated endogenous hTERT expression, contrary to transient reporters. Sp1/Sp3 expression did not correlate with hTERT promoter activity, and these TFs bound to the hTERT promoters in both telomerase-positive and telomerase-negative cells. Mutation of the proximal GC-box resulted in a dramatic decrease of hTERT promoter activity, and mutations of all five GC-boxes eliminated its transcriptional activity. Neither mutations of GC-boxes nor knockdown of endogenous Sp1 impacted promoter binding by other TFs, including E-box-binding proteins, and histone acetylation and trimethylation of histone H3K9 at the hTERT promoter in telomerase-positive and -negative cells. The result indicated that promoter binding by Sp1/Sp3 was essential, but not a limiting step, for hTERT transcription. hTERT transcription required a permissive chromatin environment. Importantly, our data also revealed different functions of GC-boxes and E-boxes in hTERT regulation; although GC-boxes were essential for promoter activity, factors bound to the E-boxes functioned to de-repress hTERT promoter.

Dual roles of c-Myc in the regulation of hTERT gene.

Human telomerase gene hTERT is important for cancer and aging. hTERT promoter is regulated by multiple transcription factors (TFs) and its activity is dependent on the chromatin environment. However, it remains unsolved how the interplay between TFs and chromatin environment controls hTERT transcription. In this study, we employed the recombinase-mediated BAC targeting and BAC recombineering techniques to dissect the functions of two proximal E-box sites at -165 and +44 nt in regulating the hTERT promoter in the native genomic contexts. Our data showed that mutations of these sites abolished promoter binding by c-Myc/Max, USF1 and USF2, decreased hTERT promoter activity, and prevented its activation by overexpressed c-Myc. Upon inhibition of histone deacetylases, mutant and wildtype promoters were induced to the same level, indicating that the E-boxes functioned to de-repress the hTERT promoter and allowed its transcription in a repressive chromatin environment. Unexpectedly, knockdown of endogenous c-Myc/Max proteins activated hTERT promoter. This activation did not require the proximal E-boxes but was accompanied by increased promoter accessibility, as indicated by augmented active histone marks and binding of multiple TFs at the promoter. Our studies demonstrated that c-Myc/Max functioned in maintaining chromatin-dependent repression of the hTERT gene in addition to activating its promoter.