Precise and Rapid Validation of Candidate Gene by Allele Specific Knockout With CRISPR/Cas9 in Wild Mice

It is a tempting goal to identify causative genes underlying phenotypic differences among inbred strains of mice, which is a huge reservoir of genetic resources to understand mammalian pathophysiology. In particular, the wild-derived mouse strains harbor enormous genetic variations that have been acquired during evolutionary divergence over 100s of 1000s of years. However, validating the genetic variation in non-classical strains was extremely difficult, until the advent of CRISPR/Cas9 genome editing tools. In this study, we first describe a T cell phenotype in both wild-derived PWD/PhJ parental mice and F1 hybrids, from a cross to C57BL/6 (B6) mice, and we isolate a genetic locus on Chr2, using linkage mapping and chromosome substitution mice. Importantly, we validate the identification of the functional gene controlling this T cell phenotype, Cd44, by allele specific knockout of the PWD copy, leaving the B6 copy completely intact. Our experiments using F1 mice with a dominant phenotype, allowed rapid validation of candidate genes by designing sgRNA PAM sequences that only target the DNA of the PWD genome. We obtained 10 animals derived from B6 eggs fertilized with PWD sperm cells which were subjected to microinjection of CRISPR/Cas9 gene targeting machinery. In the newborns of F1 hybrids, 80% (n = 10) had allele specific knockout of the candidate gene Cd44 of PWD origin, and no mice showed mistargeting of the B6 copy. In the resultant allele-specific knockout F1 mice, we observe full recovery of T cell phenotype. Therefore, our study provided a precise and rapid approach to functionally validate genes that could facilitate gene discovery in classic mouse genetics. More importantly, as we succeeded in genetic manipulation of mice, allele specific knockout could provide the possibility to inactivate disease alleles while keeping the normal allele of the gene intact in human cells

Research on pipe-following hole protection drilling technology in broken soft coal seam of the isolated island working face

It is easy to get stuck and collapse in the borehole of high stress and broken soft coal seam in the isolated island working face. This leads to great difficulty in borehole formation and poor gas extraction effect. In order to solve this problem, this paper puts forward a pipe-following borehole protection drilling technology. The 3206 isolated island working face of Wangpo Coal Mine is selected as the test site. It is analyzed that the working face needs to use high-torque and high-speed drilling rig to enhance the slag removal effect of the drilling tool and the capability to deal with accidents in the borehole. At the same time, it is necessary to consider the process of drilling wall protection in the high-stress section and the efficient slag removal process in the borehole. It is proposed to adopt the pipe-following borehole protection drilling technology in the high stress zone to achieve the effect of wall protection. The spiral drilling nitrogen-assisted slag removal process is adopted to enhance slag removal capacity and reduce the risk of coal spontaneous combustion during drilling. After the borehole passes through the high-stress area, the drilling depth of the borehole in the broken soft coal seam is further improved by optimizing the drilling tool assembly. The field test results show that the average hole depth is increased by 149% when using the second-stage hole protection drilling than when using rotary drilling directly. The average hole depth is increased by 114% when using the third-stage hole protection drilling. It shows that the pipe-following hole protection drilling is more suitable for the drilling construction of broken soft coal seam in 3206 island working face than the rotary drilling construction technology. The hole-forming rate of the plug-type screw drill pipe is higher than that of the screw thread-type screw drill pipe. The hole-forming depth of nitrogen assisted slag removal process for screw drilling is significantly greater than that of the dry screw slag removal process. \begin{document}${\text{ϕ}}$\end{document}100/63.5-28 mm plug-in sealed spiral drill pipe and nitrogen assisted slag removal process are most suitable for gas pre-extraction drilling construction in 3206 isolated island working face. The average hole depth is 100.6 m, and the hole formation rate is 80%. The gas extraction effect is better than other drilling tools and drilling slag removal technology

Spatial Heterogeneity of Typical Ecosystem Services and Their Relationships in Different Ecological–Functional Zones in Beijing–Tianjin–Hebei Region, China

Recognizing changes in ecosystem services (ES) and their relationships is the basis of achieving sustainable regional development. Regional collaborative development has become the core strategy of the development of the Beijing–Tianjin–Hebei (BTH) region. However, sub regions have different ecological changes and relationships. Here, we quantify and map ES, including water yield, sediment retention, carbon sequestration and grain productive capacity in 2000, 2005, 2010 and 2015, using several biophysical models and explore the relationships of spatial correction, trade-offs and synergies among multiple ES in different spatial scales. Results across the four years show that the quality and variation tendency of ES from each region are spatially heterogeneous. The relationship between ES that are not significant in the entire region shows different correlations in individual ecological–functional zones. From the perspective of regional disparity, the effect of land use factor and correlative mechanisms among ES are analyzed. To observe the spatiotemporal variations and relationships of ES in individual regions, land use management policies are proposed on the basis of the results of the relationships among ES

Study on Neuroendocrine-Immune Function of Cistanche deserticola and Its Rice Wine Steaming Products in Glucocorticoid-Induced Rat Model

The desert-dwelling Cistanche herb was first recorded in the “Shen Nong Herbal Classic” and is listed as the top-grade herbal medicine in this publication. The Chinese Pharmacopoeia records that pieces of Cistanche deserticola (CD) and rice wine-steamed Cistanche deserticola (WCD) can be used in the clinic as the main types of decoctions. After being steamed with rice wine, the antiaging and tonifying kidney-yang effects are enhanced. In this study, we detected the chemical content of CD and WCD and the pharmacological mechanism of invigorating kidney-yang deficiency in model rats. Aim. The purpose of this study was to examine the effects of CD and WCD on the neuroendocrine-immune function of kidney-yang deficiency in glucocorticoid-overdosed model rats. Materials and methods. Sprague Dawley (SD) rats were selected. The rats were subcutaneously injected with corticosterone water suspension for the glucocorticoid-overdosed model rats. The positive control rats were gavaged with Jinkuishenqi pills and high-, medium-, and low-dose CD/WCD suspensions (1.646 g/(kg day), 5.48 g/(kg day), 2.74 g/(kg day), and 1.37 g/(kg day), respectively); the blank control (BC) and model control (MC) groups were given the same volume of distilled water as those in the drug group for 40 consecutive days at a dose of 1 mL/100 g. After the last administration, the blood was collected from the abdominal aorta, and serum levels of T, CRH, ACTH, CORT, cortisol, IL-10, IL-6, IL-2, TNF-α, and IFN-γ were measured. Organ indexes of the thymus gland and the spleen were calculated. The expression of Bax, Bcl-2, caspase-3, Fas, and FasL in the adrenal gland was measured by immunohistochemistry. The pathological changes in the thymus gland and the adrenal gland were observed by HE staining (×200). T lymphocyte subsets in peripheral blood were detected by flow cytometry, and the expression of CaM mRNA in the hypothalamus and hypophysis tissues was also measured by RT-PCR. Results. Compared with the MC group, the CD and WCD groups exhibited increases in activity, the organ index of the thymus and the spleen, the serum levels of T, CRH, ACTH, CORT, cortisol, IL-2, and IL-10, the ratio of CD4+/CD8+, and the expression of Bcl-2, caspase-3, Fas, FasL, and CaM in the hypophysis tissue. The CD and WCD groups also exhibited reductions in the IL-6, TNF-α, and IFN-γ levels in serum and the expression of CaM mRNA in the hypothalamus. Conclusions. Each dose of CD and WCD could counteract the dysregulated sex hormone and immune factors in glucocorticoid-overdosed model rats, enhancing and restoring the effect of the hypothalamic nerve cells and improving immune function

Strong bioinspired HPA-rGO nanocomposite films via interfacial interactions for flexible supercapacitors

Flexible supercapacitors with excellent performance are needed to meet the increasing demand for wearable and flexible electronics. The challenge remains to design exceptionally flexible supercapacitors with remarkable electrochemical properties. Natural nacre shows outstanding fracture toughness due to its alternating inorganic and organic layered structure and abundant interfacial interactions, providing an inspiration for designing flexible supercapacitors. Herein, we demonstrated nacre-inspired flexible supercapacitors via synergistic interfacial interactions of π-π conjugated bonds, hydrogen bonding, and electrostatic interaction between halloysite (HA)-polyaniline (PANI) nanocomposites and graphene oxide (GO) nanosheets. The resultant nacre-inspired HPA-rGO nanocomposite films demonstrate strong tensile strength (351.9 MPa), high electrical conductivity (397.0 S cm−1), and long cycle life with ~ 85% of capacitance retention after 10,000 cycles. Furthermore, the assembled all-solid-state supercapacitors (ASSSs) based on bioinspired HPA-rGO electrodes can not only display extraordinary flexibility with no decay of capacitance behavior after 5000 bending cycles, but also deliver remarkable mass energy density up to 16.3 Wh kg−1, outperforming other flexible graphene-based supercapacitors. This nacre-inspired strategy for designing flexible electrodes provides an avenue for the next-generation power source in the fields of aerospace and smart wearable electronics

Chromosome 1 Sequence Analysis of C57BL/6J-Chr1KM Mouse Strain

The Chinese Kunming (KM) mouse is a widely used outbred mouse stock in China. However, its genetic structure remains unclear. In this study, we sequenced the genome of the C57BL/6J-Chr1KM (B6-Chr1KM) strain, the chromosome 1 (Chr 1) of which was derived from one KM mouse. With 36.6× average coverage of the entire genome, 0.48 million single nucleotide polymorphisms (SNPs) and 96,679 indels were detected on Chr 1 through comparison with reference strain C57BL/6J. Moreover, 46,590 of them were classified as novel mutations. Further functional annotation identified 155 genes harboring potentially functional variants, among which 27 genes have been associated with human diseases. We then performed sequence similarity and Bayesian concordance analysis using the SNPs identified on Chr 1 and their counterparts in three subspecies, Mus musculus domesticus, M. m. musculus, and M. m. castaneus. Both analyses suggested that the Chr 1 sequence of B6-Chr1KM was predominantly derived from M. m. domesticus while 9.7% of the sequence was found to be from M. m. musculus. In conclusion, our analysis provided a detailed description of the genetic variations on Chr 1 of B6-Chr1KM and a new perspective on the subspecies origin of KM mouse which can be used to guide further genetic studies with this mouse strain

Genome Sequencing of Chromosome 1 Substitution Lines Derived from Chinese Wild Mice Revealed a Unique Resource for Genetic Studies of Complex Traits

Mouse resources such as Collaborative Cross, outbred stocks, Hybrid Mouse Diversity Panel, and chromosome substitution strains have been instrumental to many progresses in the studies of complex traits genetics. We have established a population of chromosome 1 (Chr 1) substitution lines (C1SLs) in which donor chromosomes were derived from Chinese wild mice. Genome sequencing of 18 lines of this population showed that Chr 1 had been replaced by the donor chromosome. About 4.5 million unique single nucleotide polymorphisms and indels were discovered on Chr 1, of which 1.3 million were novel. Compared with sequenced classical inbred strains, Chr 1 of each C1SL had fivefold more variants, and more loss of function and potentially regulatory variants. Further haplotype analysis showed that the donor chromosome accumulated more historical recombination events, with the largest haplotype block being only 100 kb, and about 57% of the blocks were <1 kb. Subspecies origin analysis showed that these chromosomes had a mosaic genome structure that dominantly originated from Mus musculus musculus and M. m. castaneus subspecies, except for the C57BL/6J-Chr1KM line from M. m. domesticus. In addition, phenotyping four of these lines on blood biochemistry suggested that there were substantial phenotypic variations among our lines, especially line C57BL/6J-Chr1HZ and donor strain C57BL/6J. Further gene ontology enrichment revealed that the differentially expressed genes among liver-expressed genes between C57BL/6J and C57BL/6J-Chr1HZ were enriched in lipid metabolism biological processes. All these characteristics enable C1SLs to be a unique resource for identifying and fine mapping quantitative trait loci on mouse Chr 1, and carrying out systems genetics studies of complex traits

Loss of natural resistance to schistosome in T cell deficient rat.

Schistosomiasis is among the major neglected tropical diseases and effective prevention by boosting the immune system is still not available. T cells are key cellular components governing adaptive immune response to various infections. While common laboratory mice, such as C57BL/6, are highly susceptible to schistosomiasis, the SD rats are extremely resistant. However, whether adaptive immunity is necessary for such natural resistance to schistosomiasis in rats remains to be determined. Therefore, it is necessary to establish genetic model deficient in T cells and adaptive immunity on the resistant SD background, and to characterize liver pathology during schistosomiasis. In this study we compared experimental schistosomiasis in highly susceptible C57BL/6 (B6) mice and in resistant SD rats, using cercariae of Schistosoma japonicum. We observed a marked T cell expansion in the spleen of infected B6 mice, but not resistant SD rats. Interestingly, CD3e-/- B6 mice in which T cells are completely absent, the infectious burden of adult worms was significantly higher than that in WT mice, suggesting an anti-parasitic role for T cells in B6 mice during schistosome infection. In further experiments, we established Lck deficient SD rats by using CRISPR/Cas9 in which T cell development was completely abolished. Strikingly, we found that such Lck deficiency in SD rats severely impaired their natural resistance to schistosome infection, and fostered parasite growth. Together with an additional genetic model deficient in T cells, the CD3e-/- SD rats, we confirmed the absence of T cell resulted in loss of natural resistance to schistosome infection, but also mitigated liver immunopathology. Our further experiments showed that regulatory T cell differentiation in infected SD rats was significantly decreased during schistosomiasis, in contrast to significant increase of regulatory T cells in infected B6 mice. These data suggest that T cell mediated immune tolerance facilitates persistent infection in mice but not in SD rats. The demonstration of an important role for T cells in natural resistance of SD rats to schistosomiasis provides experimental evidences supporting the rationale to boost T cell responses in humans to prevent and treat schistosomiasis

Ultratough graphene-black phosphorus films

Graphene-based films with high toughness have many promising applications, especially for flexible energy storage and portable electrical devices. Achieving such high-toughness films, however, remains a challenge. The conventional mechanisms for improving toughness are crack arrest or plastic deformation. Herein we demonstrate black phosphorus (BP) functionalized graphene films with record toughness by combining crack arrest and plastic deformation. The formation of covalent bonding P-O-C between BP and graphene oxide (GO) nanosheets not only reduces the voids of GO film but also improves the alignment degree of GO nanosheets, resulting in high compactness of the GO film. After further chemical reduction and p-p stacking interactions by conjugated molecules, the alignment degree of rGO nanosheets was further improved, and the voids in lamellar graphene film were also further reduced. Then, the compactness of the resultant graphene films and the alignment degree of reduced graphene oxide nanosheets are further improved. The toughness of the graphene film reaches as high as similar to 51.8 MJ m(-3), the highest recorded to date. In situ Raman spectra and molecular dynamics simulations reveal that the record toughness is due to synergistic interactions of lubrication of BP nanosheets, P-O-C covalent bonding, and p-p stacking interactions in the resultant graphene films. Our tough black phosphorus functionalized graphene films with high tensile strength and excellent conductivity also exhibit high ambient stability and electromagnetic shielding performance. Furthermore, a supercapacitor based on the tough films demonstrated high performance and remarkable flexibility