Molecular and Evolutionary Analysis of RNA-Protein Interactions in Telomerase Regulation.

Telomerase is an enzyme involved in the maintenance of telomeres. Telomere shortening due to the end-replication problem is a threat to the genome integrity of all eukaryotes. Telomerase inside cells depends on a myriad of protein-protein and RNA-protein interactions to properly assemble and regulate the function of the telomerase holoenzyme. These interactions are well studied in model eukaryotes, like humans, yeast, and the ciliated protozoan known as Tetrahymena thermophila. Emerging evidence also suggests that deep-branching eukaryotes, such as the parasitic protist Trypanosoma brucei require conserved and novel RNA-binding proteins for the assembly and function of their telomerase. In this review, we will discuss telomerase regulatory pathways in the context of telomerase-interacting proteins, with special attention paid to RNA-binding proteins. We will discuss these interactors on an evolutionary scale, from parasitic protists to humans, to provide a broader perspective on the extensive role that protein-protein and RNA-protein interactions play in regulating telomerase activity in eukaryotes.

Proteomic analysis defines the interactome of telomerase in the protozoan parasite, Trypanosoma brucei.

Telomerase is a ribonucleoprotein enzyme responsible for maintaining the telomeric end of the chromosome. The telomerase enzyme requires two main components to function: the telomerase reverse transcriptase (TERT) and the telomerase RNA (TR), which provides the template for telomeric DNA synthesis. TR is a long non-coding RNA, which forms the basis of a large structural scaffold upon which many accessory proteins can bind and form the complete telomerase holoenzyme. These accessory protein interactions are required for telomerase activity and regulation inside cells. The interacting partners of TERT have been well studied in yeast, human, and Tetrahymena models, but not in parasitic protozoa, including clinically relevant human parasites. Here, using the protozoan parasite, Trypanosoma brucei (T. brucei) as a model, we have identified the interactome of T. brucei TERT (TbTERT) using a mass spectrometry-based approach. We identified previously known and unknown interacting factors of TbTERT, highlighting unique features of T. brucei telomerase biology. These unique interactions with TbTERT, suggest mechanistic differences in telomere maintenance between T. brucei and other eukaryotes.

Discriminatory Value of the Ascending Aorta Diameter in Suspected Acute Type A Aortic Dissection.

OBJECTIVE: The objective was to determine if ascending aorta (AscAo) diameters measured by noncontrast computed tomography (CT) allow for meaningful discrimination between patients with and without type A aortic dissection (TAAD), ideally with 100% sensitivity. METHODS: This study was a retrospective analysis of cases of TAAD, as well as controls, undergoing evaluation for TAAD with CT aortography, presenting to 21 emergency departments within an integrated health system between 2007 and 2015. AscAo diameters were determined using axial noncontrast CT images at the level of the right main pulmonary artery by two readers. AscAo diameters were additionally normalized for age, sex, and body surface area (assessed by a Z-score, which is the number of standard deviations between the observed and expected AscAo diameters). Overall model discrimination was assessed using the area under the receiver operating characteristic curve (AUC). Comparative discrimination was assessed using both the change in AUC (∆AUC) and the continuous net reclassification index (NRI). RESULTS: A total of 230 cases of TAAD and 325 controls were included in the study. The median ages for cases and controls were 65 and 62 years, and the median AscAo diameters were 50 and 35 mm, respectively. The raw and normalized AscAo diameters demonstrated similarly excellent discrimination (AUCs of 0.96 vs. 0.97, respectively; ∆AUC = 0.01, p = 0.09) and an NRI of 0.30 (95% confidence interval [CI] = 0.13-0.47), both indicating small incremental improvements in classification with the use of the normalized AscAo measures. A raw AscAo diameter of 34 mm and a normalized Z-score of 1.84 both yielded 100% sensitivity for TAAD, with respective specificities of 35% (95% CI = 29.6%-40.2%) and 67% (95% CI = 61.7%-72.2%). CONCLUSIONS: Nearly all patients with TAAD appear to have enlarged AscAo diameters as measured by noncontrast CT, whereas most patients with suspected but absent TAAD have relatively normal AscAo diameters. Both raw and normalized AscAo measures provided relatively comparable discriminatory value. If validated, these data may be useful in adjudicating risk among patients with suspected TAAD in whom a criterion standard test is unavailable, nondiagnostic, or contraindicated.

Effect of Glucose on 3D Cardiac Microtissues Derived from Human Induced Pluripotent Stem Cells.

Maternal hyperglycemia is a risk factor for fetal cardiac anomalies. This study aimed to assess the effect of high glucose on human induced pluripotent stem cell-derived cardiomyocyte self-assembly into 3D microtissues and their calcium handling. Stem cells were differentiated to beating cardiomyocytes using established protocols. On the final day of the differentiation process, cells were treated with control media, 12 mM glucose, or 12 mM mannitol (an osmolality control). Once beating, the cardiac cells were dissociated with trypsin, collected, mixed with collagen, and plated into custom-made silicone micro molds in order to generate 3D cardiac microtissues. A time-lapse microscope took pictures every 4 h to quantify the kinetics of cellular self-assembly of 3D cardiac tissues. Fiber widths were recorded at 4-h intervals and plotted over time to assess cardiomyocyte 3D fiber self-assembly. Microtissue calcium flux was recorded with optical mapping by pacing microtissues at 0.5 and 1.0 Hz. Exposure to high glucose impaired the ability of cardiomyocytes to self-assemble into compact microtissues, but not their ability to spontaneously contract. Glucose-exposed cardiomyocytes took longer to self-assemble and finished as thicker fibers. When cardiac microtissues were paced at 0.5 and 1.0 Hz, those exposed to high glucose had altered calcium handling with shorter calcium transient durations, but larger amplitudes of the calcium transient when compared to controls. Additional studies are needed to elucidate a potential mechanism for these findings. This model provides a novel method to assess the effects of exposures on the cardiomyocytes' intrinsic abilities for organogenesis in 3D.