Evolution of Helix Formation in the Ribosomal Internal Transcribed Spacer 2 (ITS2) and Its Significance for RNA Secondary Structures

Helices are the most common elements of RNA secondary structure. Despite intensive investigations of various types of RNAs, the evolutionary history of the formation of new helices (novel helical structures) remains largely elusive. Here, by studying the nuclear ribosomal Internal Transcribed Spacer 2 (ITS2), a fast-evolving part of the eukaryotic nuclear ribosomal operon, we identify two possible types of helix formation: one type is “dichotomous helix formation”—transition from one large helix to two smaller helices by invagination of the apical part of a helix, which significantly changes the shape of the original secondary structure but does not increase its complexity (i.e., the total length of the RNA). An alternative type is “lateral helix formation”—origin of an extra helical region by the extension of a bulge loop or a spacer in a multi-helix loop of the original helix, which does not disrupt the pre-existing structure but increases RNA size. Moreover, we present examples from the RNA sequence literature indicating that both types of helix formation may have implications for RNA evolution beyond ITS2.     

Multistep Photoluminescence Decay Reveals Dissociation of Geminate Charge Pairs in Organolead Trihalide Perovskites

Charge carrier dynamics in organolead iodide perovskites is analyzed by employing time‐resolved photoluminescence spectroscopy with several ps time resolution. The measurements performed by varying photoexcitation intensity over five orders of magnitude enable separation of photoluminescence components related to geminate and nongeminate charge carrier recombination and to address the dynamics of an isolated geminate electron–hole pair. Geminate recombination dominates at low excitation fluence and determines the initial photoluminescence decay. This decay component is remarkably independent of the material structure and experimental conditions. It is demonstrated that dependences of the geminate and nongeminate radiative recombination components on excitation intensity, repetition rate, and temperature, are hardly compatible with carrier trapping and exciton dissociation models. On the basis of semiclassical and quantum mechanical numerical calculation results, it is argued that the fast photoluminescence decay originates from gradual spatial separation of photogenerated weakly bound geminate charge pairs.     

Identification of cardiac stem cells within mature cardiac myocytes

Cardiac stem cells are described in a number of mammalian species including humans. Cardiac stem cell clusters consisting of both lineage-negative and partially committed cells are generally identified between contracting cardiac myocytes. In the present study, c-kit⁺, Sca⁺, and Isl1⁺ stem cells were revealed to be located inside the sarcoplasm of cardiac myocytes in myocardial cell cultures derived from newborn, 20-, and 40-day-old rats. Intracellularly localized cardiac stem cells had a coating or capsule with a few pores that opened into the host cell sarcoplasm. The similar structures were also identified in the suspension of freshly isolated myocardial cells (ex vivo) of 20- and 40-day-old rats. The results from this study provide direct evidence for the replicative division of encapsulated stem cells, followed by their partial cardiomyogenic differentiation. The latter is substantiated by the release of multiple transient amplifying cells following the capsule rupture. In conclusion, functional cardiac stem cells can reside not only exterior to but also within cardiomyocytes.     

Distinctive roles of receptor‐interacting protein kinases 1 and 3 in caspase‐independent cell death of L929

Upon tumour necrosis factor alpha (TNFα) stimulation, cells respond actively by way of cell survival, apoptosis or programmed necrosis. The receptor‐interacting proteins 1 (RIP1) and 3 (RIP3) are responsible for TNFα‐mediated programmed necrosis. To delineate the differential contributions of RIP3 and RIP1 to programmed necrosis, L929 cells were stimulated with TNFα, carbobenzoxy‐valyl‐alanyl‐aspartyl‐[O‐methyl]‐fluoromethylketone (zVAD) or zVAD along with TNFα following RNA interference against RIP1 and RIP3, respectively. RIP1 silencing did not protect cells from TNFα‐mediated cell death, while RIP3 down‐regulation made them refractory to TNFα. The heat shock protein 90 inhibitor geldanamycin (GA) down‐regulated both RIP1 and RIP3 expression, which rendered cells resistant to zVAD/TNFα‐mediated cell death but not to TNFα‐mediated cell death alone. Therefore, the protective effect of GA on zVAD/TNFα‐stimulated necrosis might be attributed to RIP3, not RIP1, down‐regulation. Pretreatment of L929 cells with rapamycin mitigated zVAD‐mediated cell death, while the autophagy inhibitor chloroquine did not affect necrotic cell death. Meanwhile, necrotic cell death by zVAD and TNFα was caused by reactive oxygen species generation and effectively diminished by lipid‐soluble butylated hydroxyanisole. Taken together, the results indicate that RIP1 and RIP3 can independently mediate death signals being transduced by two different death stimuli, zVAD and TNFα. Copyright © 2013 John Wiley & Sons, Ltd.     

Reliably Detecting Clinically Important Variants Requires Both Combined Variant Calls and Optimized Filtering Strategies

A diversity of tools is available for identification of variants from genome sequence data. Given the current complexity of incorporating external software into a genome analysis infrastructure, a tendency exists to rely on the results from a single tool alone. The quality of the output variant calls is highly variable however, depending on factors such as sequence library quality as well as the choice of short-read aligner, variant caller, and variant caller filtering strategy. Here we present a two-part study first using the high quality ‘genome in a bottle’ reference set to demonstrate the significant impact the choice of aligner, variant caller, and variant caller filtering strategy has on overall variant call quality and further how certain variant callers outperform others with increased sample contamination, an important consideration when analyzing sequenced cancer samples. This analysis confirms previous work showing that combining variant calls of multiple tools results in the best quality resultant variant set, for either specificity or sensitivity, depending on whether the intersection or union, of all variant calls is used respectively. Second, we analyze a melanoma cell line derived from a control lymphocyte sample to determine whether software choices affect the detection of clinically important melanoma risk-factor variants finding that only one of the three such variants is unanimously detected under all conditions. Finally, we describe a cogent strategy for implementing a clinical variant detection pipeline; a strategy that requires careful software selection, variant caller filtering optimizing, and combined variant calls in order to effectively minimize false negative variants. While implementing such features represents an increase in complexity and computation the results offer indisputable improvements in data quality.     

Relationship between Initial Telomere Length,Initial Telomerase Activity,Age, and Replicative Capacity of Nucleus Pulposus Chondrocytes in Human Intervertebral Discs: What Is a Predictor of Replicative Potential?

There is evidence that telomere length (TL), telomerase activity (TA), and age are related to the replicative potential of human nucleus pulposus chondrocytes (NPCs). However, it has not yet been established if any of these factors can serve as predictors of the replicative potential of NPCs. To establish predictors of the replicative potential of NPCs, we evaluated potential relationships between replicative capacity of NPCs, initial TL (telomere length at the first passage), initial TA (telomerase activity at the first passage), and age. Nucleus pulposus specimens were obtained from 14 patients of various ages undergoing discectomy. NPCs were serially cultivated until the end of their replicative lifespans. Relationships among cumulative population doubling level (PDL), initial TL, initial TA, and age were analyzed. Initial TA was negatively correlated with age (r = -0.674, P = 0.008). However, no correlation between initial TL and age was observed. Cumulative PDL was also negatively correlated with age (r = -0.585, P = 0.028). Although the cumulative PDL appeared to increase with initial TL or initial TA, this trend was not statistically significant. In conclusion, age is the sole predictor of the replicative potential of human NPCs, and replicative potential decreases with age. Initial TL and initial TA are not predictors of replicative potential, and can serve only as reference values.     

The Consequences of Precipitation Seasonality for Mediterranean-Ecosystem Vegetation of South Africa

Globally, mediterranean-climate ecosystem vegetation has converged on an evergreen, sclerophyllous and shrubby growth form. The particular aspects of mediterranean-climate regions that contribute to this convergence include summer droughts and relatively nutrient-poor soils. We hypothesised that winter-precipitation implies stressful summer droughts and leaches soils due to greater water availability (i.e. balance between precipitation and potential evapotranspiration; P–PET) during cold periods. We conducted a comparative analysis of normalised difference vegetation indices (NDVI) and edaphic and climate properties across the biomes of South Africa. NDVI was strongly correlated with both precipitation and P–PET (r² = 0.8). There was no evidence, however, that winter-precipitation reduces NDVI in comparison to similar amounts of summer-precipitation. Base saturation (BS), a measure of soil leaching was, however, negatively related to P–PET (r² = 0.64). This led to an interaction between P–PET and BS in determining NDVI, indicating the existence of a trade-off between water availability and soil nutrients that enables NDVI to increase with precipitation, despite negative consequences for soil nutrient availability. The mechanism of this trade-off is suggested to be that water increases nutrient accessibility. This implies that along with nutrient-depauperate geologies and long periods of time since glaciation, the winter-precipitation may have contributed to the highly leached status of the soils. Since many of the ecophysiological characteristics of mediterranean-ecosystem flora are associated with low nutrient availabilities (e.g. evergreen foliage, sclerophylly, cluster roots), we conclude that mediterranean-climates promote convergence of growth-forms in these regions through high leaching capacity.     

Evidence for K+ channel control in Vicia guard cells coupled by G-proteins to a 7TMS receptor mimetic

To explore the involvement of a class of seven-trans-membrane-span (7TMS) receptors in cellular signalling, a synthetic analogue (mas7) of the amphipathic tetradecapeptide mastoparan was used to mimic hormonal stimulus in guard cells of Vicia faba. The ability for mas7 to substitute for an activated receptor complex was assayed by the effect on guard cell ion channel activities in the absence of any hormonal stimulus. Currents carried by inward-(I_K,in) and outward-(I_K,out) rectifying potassium channels were determined under voltage clamp conditions before, during, and after exposure to mas7. The dominant effect of mas7 was to inactivate I_K,in within 30 sec of application. By contrast, I_K,out was largely unaffected under these conditions. The effect of mas7 on I_K,in was both concentration- and voltage-dependent. At any one clamp voltage, mas7 inactivation showed Michaelian behaviour, with a mean K_i of 0.05 ± 0.02 µM at ?240 mV. Increasing mas7 concentration also shifted the voltage for half-maximal activation of the current negative, with 0.5 µM mas7 effecting a ?13 ± 2 mV displacement and lengthening the halftime for activation of the current by up to threefold. By contrast, the non-amphipathic analogue of mas7, masCP, had no appreciable effect on the steady-state current or its activation kinetics; nor was the poly-cation polylysine able to substitute for mas7 in its action on the K⁺ channels. Application of the non-hydrolysable analogue of GDP, GDP-β-S, either by iontophoresis or by diffusion from the microelectrode, effectively blocked mas7-induced inactivation of I_K,in. These, and additional results provide in vivo evidence for the involvement of G-protein-linked 7TMS receptors in the regulation of membrane transport in a higher plant cell.