Specialized RNA endonucleases are critical for efficient activity of the CRISPR-Cas defense mechanisms against invading DNA or RNA. Cas6-type enzymes are the RNA endonucleases in many type I and type ...III CRISPR-Cas systems. These enzymes are diverse and critical residues involved in the recognition and cleavage of RNA substrates are not universally conserved. Cas6 endonucleases associated with the CRISPR-Cas subtypes I-A, I-B, I-C, I-E and I-F, as well as III-B have been studied from three archaea and four bacteria thus far. However, until now information about subtype I-D specific Cas6 endonucleases has remained scarce. Here, we report the biochemical analysis of Cas6-1, which is specific for the crRNA maturation from the subtype I-D CRISPR-Cas system of Synechocystis sp. PCC 6803. Assays of turnover kinetics suggest a single turnover mechanism for Cas6-1. The mutation of conserved amino acids R29A, H32A-S33A and H51A revealed these as essential, whereas the parallel mutation of R175A-R176A led to a pronounced and the K155A mutation to a slight reduction in enzymatic activity. In contrast, the mutations R67A, R81A and K231A left the enzymatic activity unchanged. These results are in accordance with the predominant role of histidine residues in the active site and of positively charged residues in RNA binding. Nevertheless, the protein-RNA interaction site seems to differ from other known systems, since imidazole could not restore the mutated histidine site.
Mass movements play an important role in landscape evolution of high mountain areas such as the Himalayas. Yet, establishing numerical age control and reconstructing transport dynamics of past events ...is challenging. To fill this research gap, we bring luminescence dating to the test in an extremely challenging environment: the Pokhara Valley in Nepal. This is challenging for two reasons: (i) the optically stimulated luminescence (OSL) sensitivity of quartz, typically the mineral of choice for dating sediments younger than 100 ka, is poor, and (ii) highly rapid and turbid conditions during mass movement transport hamper sufficient OSL signal resetting prior to deposition, which eventually results in age overestimation. Here, we first assess the applicability of single-grain feldspar dating of medieval mass movement deposits catastrophically emplaced in the Pokhara Valley. Second, we exploit the poor bleaching mechanisms to get insight into the sediment dynamics of this paleo-mass movement through bleaching proxies. The Pokhara Valley is a unique setting for our case study, considering the availability of an extensive independent radiocarbon dataset as a geochronological benchmark. Single-grain infrared stimulated luminescence (IRSL) signals were measured at 50 ∘C (IRSL-50) and post-infrared infrared stimulated luminescence signals at 150 ∘C (pIRIR-150). Our results show that the IRSL-50 signal is better bleached than the pIRIR-150 signal. A bootstrapped minimum age model (bMAM) is applied to retrieve the youngest subpopulation to estimate the paleodose. However, burial ages calculated with this paleodose overestimate the radiocarbon ages by an average factor of ∼23 (IRSL-50) and ∼72 (pIRIR-150), showing that dating of the Pokhara Formation with a single-grain approach was not successful for most samples. Some samples, however, only slightly overestimate the true emplacement age and thus could be used for a rough age estimation. Large inheritances in combination with the scatter in the single-grain dose distributions show that the sediments have been transported under extremely limited light exposure prior to deposition, which is consistent with the highly turbid nature of the sediment-laden flood and debris flows depositing the Pokhara gravels. To investigate the sediment transport dynamics in more detail, we studied three bleaching proxies: the percentage of grains in saturation 2D0 criteria, the percentage of best-bleached grains ( 2σ range of bMAM- De ) and the overdispersion (OD). None of the three bleaching proxies indicate a spatial relationship with runout distance of the mass movement deposits. We interpret this as evidence for the lack of bleaching during transport, which reflects the catastrophic nature of the event. While not providing reliable burial ages of the Pokhara mass movement deposits, single-grain feldspar dating can potentially be used as an age range finder method. Our approach shows the potential of luminescence techniques to provide insights in sediment transport dynamics of extreme and rare mass movement events in mountainous regions.
Nitrate is an abundant nutrient and electron acceptor throughout Earth's biosphere. Virtually all nitrate in nature is produced by the oxidation of nitrite by the nitrite oxidoreductase (NXR) ...multiprotein complex. NXR is a crucial enzyme in the global biological nitrogen cycle, and is found in nitrite-oxidizing bacteria (including comammox organisms), which generate the bulk of the nitrate in the environment, and in anaerobic ammonium-oxidizing (anammox) bacteria which produce half of the dinitrogen gas in our atmosphere. However, despite its central role in biology and decades of intense study, no structural information on NXR is available. Here, we present a structural and biochemical analysis of the NXR from the anammox bacterium Kuenenia stuttgartiensis, integrating X-ray crystallography, cryo-electron tomography, helical reconstruction cryo-electron microscopy, interaction and reconstitution studies and enzyme kinetics. We find that NXR catalyses both nitrite oxidation and nitrate reduction, and show that in the cell, NXR is arranged in tubules several hundred nanometres long. We reveal the tubule architecture and show that tubule formation is induced by a previously unidentified, haem-containing subunit, NXR-T. The results also reveal unexpected features in the active site of the enzyme, an unusual cofactor coordination in the protein's electron transport chain, and elucidate the electron transfer pathways within the complex.
Malignant rhabdoid tumors (MRTs) are aggressive pediatric cancers arising in brain, kidney and soft tissues, which are characterized by loss of the tumor suppressor SNF5/SMARCB1. MRTs are poorly ...responsive to chemotherapy and thus a high unmet clinical need exists for novel therapies for MRT patients. SNF5 is a core subunit of the SWI/SNF chromatin remodeling complex which affects gene expression by nucleosome remodeling. Here, we report that loss of SNF5 function correlates with increased expression of fibroblast growth factor receptors (FGFRs) in MRT cell lines and primary tumors and that re-expression of SNF5 in MRT cells causes a marked repression of FGFR expression. Conversely, siRNA-mediated impairment of SWI/SNF function leads to elevated levels of FGFR2 in human fibroblasts. In vivo, treatment with NVP-BGJ398, a selective FGFR inhibitor, blocks progression of a murine MRT model. Hence, we identify FGFR signaling as an aberrantly activated oncogenic pathway in MRTs and propose pharmacological inhibition of FGFRs as a potential novel clinical therapy for MRTs.
Highlights • Spinal GLP-1 axons target primarily sympathetic preganglionic neurons. • Spinal GLP-1 axons innervate interneurons that may regulate sympathetic outflow. • Many GLP-1 neurons in the ...medulla are spinally-projecting. • The lumbar cord contains YFP-expressing neurons that do not innervate the brain.
We cloned two β subunits of large-conductance calcium-activated potassium (BK) channels, hKCNMB3 (BKβ1) and hKCNMB4 (BKβ4). Profiling mRNA expression showed that hKCNMB3 expression is enriched in ...testis and hKCNMB4 expression is very prominent in brain. We coexpressed BK channel α (BKα) and BKβ4 subunits in vitro in CHO cells. We compared BKα/β4 mediated currents with those of smooth muscle BKα/β1 channels. BKβ4 slowed activation kinetics more significantly, led to a steeper apparent calcium sensitivity, and shifted the voltage range of BK current activation to more negative potentials than BKβ1. BKα/β4 channels were not blocked by 100 nM charybdotoxin or iberiotoxin, and were activated by 17β-estradiol.
Previous research demonstrates that listeners perceive women’s voices as more attractive when recorded at high compared to low fertility phases of the menstrual cycle. This effect has been repeated ...with multiple voice recording samples, but one stimuli set has shown particularly robust replications. First collected by Pipitone and Gallup (2008), women were recorded counting from 1–10 on approximately the same day and time once a week for 4 weeks. Repeatedly, studies using these recordings have shown that naturally cycling women recorded at high fertility are rated as more attractive compared to voices of the same women at low fertility. Additionally, these stimuli have been shown to elicit autonomic nervous system arousal and precipitate a rise in testosterone levels among listeners. Although previous studies have examined the acoustic properties of voices across the menstrual cycle, they reach little consensus. The current study evaluates Pipitone and Gallup’s voice stimuli from an acoustic perspective, analyzing specific vocal characteristics of both naturally cycling women and women taking hormonal contraceptives. Results show that among naturally cycling women, variation in vocal amplitude (shimmer) was significantly lower in high fertility recordings compared to the women’s voices at low fertility. Harmonics-to-noise ratio and variation in voice pitch (jitter) also fluctuated systematically across voices sampled at different times during the menstrual cycle, though these effects were not statistically significant. It is possible that these acoustic changes could account for some of the replicated perceptual, hormonal, and physiological changes documented in prior literature using these voice stimuli.
Applied geochemistry and environmental sciences invariably deal with compositional data. Classically, the original or log-transformed absolute element concentrations are studied. However, ...compositional data do not vary independently, and a concentration based approach to data analysis can lead to faulty conclusions. For this reason a better statistical approach was introduced in the 1980s, exclusively based on relative information. Because the difference between the two methods should be most pronounced in large-scale, and therefore highly variable, datasets, here a new dataset of agricultural soils, covering all of Europe (5.6millionkm2) at an average sampling density of 1site/2500km2, is used to demonstrate and compare both approaches. Absolute element concentrations are certainly of interest in a variety of applications and can be provided in tabulations or concentration maps. Maps for the opened data (ratios to other elements) provide more specific additional information. For compositional data XY plots for raw or log-transformed data should only be used with care in an exploratory data analysis (EDA) sense, to detect unusual data behaviour, candidate subgroups of samples, or to compare pre-defined groups of samples. Correlation analysis and the Euclidean distance are not mathematically meaningful concepts for this data type. Element relationships have to be investigated via a stability measure of the (log-)ratios of elements. Logratios are also the key ingredient for an appropriate multivariate analysis of compositional data.
► Major element concentrations in agricultural soils of Europe are presented. ► Results of classical data analysis versus compositional data analysis are compared. ► Both approaches deliver important and complementary results. ► With compositional data XY diagrams should not be used to discuss correlation. ► Multivariate data analysis of compositional data should be based on logratio-transformed data.
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