Structure and mechanism of the CMR complex for CRISPR-Mediated antiviral immunity

The prokaryotic clusters of regularly interspaced palindromic repeats (CRISPR) system utilizes genomically encoded CRISPR RNA (crRNA), derived from invading viruses and incorporated into ribonucleoprotein complexes with CRISPR-associated (CAS) proteins, to target and degrade viral DNA or RNA on subsequent infection. RNA is targeted by the CMR complex. In Sulfolobus solfataricus, this complex is composed of seven CAS protein subunits (Cmr1-7) and carries a diverse "payload" of targeting crRNA. The crystal structure of Cmr7 and low-resolution structure of the complex are presented. S. solfataricus CMR cleaves RNA targets in an endo-nucleolytic reaction at UA dinucleotides. This activity is dependent on the 8 nt repeat-derived 5' sequence in the crRNA, but not on the presence of a proto-spacer-associated motif (PAM) in the target. Both target and guide RNAs can be cleaved, although a single molecule of guide RNA can support the degradation of multiple targets.Publisher PDFPeer reviewe

Ex vivo Dynamics of Human Glioblastoma Cells in a Microvasculature-on-a-Chip System Correlates with Tumor Heterogeneity and Subtypes

The perivascular niche (PVN) plays an essential role in brain tumor stem-like cell (BTSC) fate control, tumor invasion, and therapeutic resistance. Here, a microvasculature-on-a-chip system as a PVN model is used to evaluate the ex vivo dynamics of BTSCs from ten glioblastoma patients. BTSCs are found to preferentially localize in the perivascular zone, where they exhibit either the lowest motility, as in quiescent cells, or the highest motility, as in the invasive phenotype, with migration over long distance. These results indicate that PVN is a niche for BTSCs, while the microvascular tracks may serve as a path for tumor cell migration. The degree of colocalization between tumor cells and microvessels varies significantly across patients. To validate these results, single-cell transcriptome sequencing (10 patients and 21 750 single cells in total) is performed to identify tumor cell subtypes. The colocalization coefficient is found to positively correlate with proneural (stem-like) or mesenchymal (invasive) but not classical (proliferative) tumor cells. Furthermore, a gene signature profile including PDGFRA correlates strongly with the “homing” of tumor cells to the PVN. These findings demonstrate that the model can recapitulate in vivo tumor cell dynamics and heterogeneity, representing a new route to study patient-specific tumor cell functions

Effects of Confining Pressure and Hydrostatic Pressure on the Fracturing of Rock under Cyclic Electrohydraulic Shock Waves

For an array of applications of the high voltage pulse discharge technology in reservoir stimulations and to gain a deeper understanding of the fractures mechanism of deep well rock under cyclic electrohydraulic shock waves (EHSWs), the effect of confining pressure and hydrostatic pressure on the fracturing of rock under EHSWs are investigated in this paper. Firstly, a two-dimensional (2D) water-explosive numerical model is built to match the computed peak pressure of the EHSW with that obtained by the empirical formula by tuning the relevant parameters, based on the equivalent method of EHSWs. Then, a rock model is established to obtain the stress distribution under static loads. Subsequently, the water-explosive model is coupled with the rock model to obtain the stress distribution under static and dynamic loads. In addition, based on this coupling model, the influences of confining pressure and hydrostatic pressure on circumferential stress, radial stress in the rock and the fracturing of rock around the wellbore are discussed. Finally, two improvement measures (increasing discharge energy and changing loading mode) are proposed to acquire greater fracture density based on intensive numerical simulations. The results show that the increase in hydrostatic pressure is beneficial to the crack formation and development, whereas confining pressure is harmful. Moreover, the inhibitory effect of confining pressure on crack formation is greater than the promotion effect of hydrostatic pressure on crack formation. Increasing the discharge energy can effectively promote the development of the number and length of main cracks. Under four repetitive loading modes with the same total discharge energy (1.36 × 15 kJ), the greatest fracture density can be obtained by using repetitive loading mode with a gradually decreasing mode of discharge energy (first level: 2 times (1.36 × 5 kJ); second level: 5 times (1.36 × 1 kJ))

Multiplexed, Sequential Secretion Analysis of the Same Single Cells Reveals Distinct Effector Response Dynamics Dependent on the Initial Basal State

The effector response of immune cells dictated by an array of secreted proteins is a highly dynamic process, requiring sequential measurement of all relevant proteins from single cells. Herein, a microchip-based, 10-plexed, sequential secretion assay on the same single cells and at the scale of approximate to 5000 single cells measured simultaneously over 4 time points are shown. It is applied to investigating the time course of single human macrophage response to toll-like receptor 4 (TLR4) ligand lipopolysaccharide (LPS) and reveals four distinct activation modes for different proteins in single cells. Protein secretion dynamics classifies the cells into two major activation states dependent on the basal state of each cell. Single-cell RNA sequencing performed on the same samples at the matched time points further demonstrates the existence of two major activation states at the transcriptional level, which are enriched for translation versus inflammatory programs, respectively. These results show a cell-intrinsic heterogeneous response in a phenotypically homogeneous cell population. This work demonstrates the longitudinal tracking of protein secretion signature in thousands of single cells at multiple time points, providing dynamic information to better understand how individual immune cells react to pathogenic challenges over time and how they together constitute a population response

Reduced Carbon Dioxide Sink and Methane Source under Extreme Drought Condition in an Alpine Peatland

Potential changes in both the intensity and frequency of extreme drought events are vital aspects of regional climate change that can alter the distribution and dynamics of water availability and subsequently affect carbon cycles at the ecosystem level. The effects of extreme drought events on the carbon budget of peatland in the Zoige plateau and its response mechanisms were studied using an in-field controlled experimental method. The results indicated that the peatland ecosystem of the Zoige plateau functioned as a carbon sink while under the control (CK) or extreme drought (D) treatment throughout the entire growing season. Maximum fluxes of methane (CH4) emissions and the weakest carbon sink activity from this ecosystem were in the early growth stage, the most powerful carbon sink activity was during the peak growth stage, while the absorption sink activity of carbon dioxide (CO2) and CH4 was present during the senescence stage. Extreme drought reduced the gross primary productivity (GPP) and ecosystem respiration (Re) of the peatland ecosystem by 14.5% and 12.6%, respectively (p < 0.05) and the net ability to store carbon was reduced by 11.3%. Overall, the GPP was highly sensitive to extreme drought. Moreover, extreme drought significantly reduced the CH4 fluxes of the ecosystem and even changed the peatland from a CH4 emission source to a CH4 sink. Subsequent to drought treatment, extreme drought was also shown to have a carry-over effect on the carbon budget of this ecosystem. Soil water content and soil temperature were the main driving factors of carbon budget change in the peatland of the Zoige plateau, but with the increase in soil depth, these driving forces were decreased. The findings indicated that frequent extreme drought events in the future might reduce the net carbon sink function of peatland areas, with an especially strong influence on CO2

Spatial patterns of leaf C-13 and its relationship with plant functional groups and environmental factors in China

The leaf carbon isotope ratio (C-13) is a useful parameter for predicting a plant's water use efficiency, as an indicator for plant classification, and even in the reconstruction of paleoclimatic environments. In this study, we investigated the spatial pattern of leaf C-13 values and its relationship with plant functional groups and environmental factors throughout China. The high leaf C-13 in the database appeared in central and western China, and the averaged leaf C-13 was -27.15, with a range from -21.05 to -31.5. The order of the averaged C-13 for plant life forms from most positive to most negative was subshrubs>herbs=shrubs>trees>subtrees. Leaf C-13 is also influenced by some environmental factors, such as mean annual precipitation, relative humidity, mean annual temperature, solar hours, and altitude, although the overall influences are still relatively weak, in particular the influence of MAT and altitude. And we further found that plant functional types are dominant factors that regulate the magnitude of leaf C-13 for an individual site, whereas environmental conditions are key to understanding spatial patterns of leaf C-13 when we consider China as a whole. Ultimately, we conducted a multiple regression model of leaf C-13 with environmental factors and mapped the spatial distribution of leaf C-13 in China by using this model. However, this partial least squares model overestimated leaf C-13 for most life forms, especially for deciduous trees, evergreen shrubs, and subtrees, and thus need more improvement in the future

Spatio-Temporal Variations and Socio-Economic Driving Forces for Wetland Area Changes: Insights from 2008–2017 Data of Yunnan Province, China

An in-depth understanding of the associations between variations in the wetland area and socio-economic driving forces is essential owing to rapid urbanization. However, to date, no study has performed a quantitative study on the relationships between spatio-temporal patterns for wetland area variations and socio-economic driving factors in Yunnan Province. Based on Statistical Yearbook data, we found that during 10 years, different types of wetlands exhibited different change rates, with obvious spatial heterogeneity. The overall increase in wetland area in Yunnan Province was 13.35%, of which the increases in river, lake, and swamp wetland areas were 46.39%, −3.12%, and 295.56%, respectively. At the city level, the maximum decrease and increase in total wetland area were noted in Xishuangbanna (−84.30%) and Diqing (+185.22%), respectively. A total of 9 of 24 factors which were further selected according to collinearity diagnostics might help interpret changes in the wetland area of Yunnan Province according to the regression analysis results (R2 = 0.749, p < 0.01). Moreover, in different city development periods, the key socio-economic factors were different, which should be considered separately when formulating policies. Our results may clarify the socio-economic influencing factors for wetland spatio-temporal changes and help to guide policymakers