CRISPR-Edited Stem Cells in a Patient with HIV and Acute Lymphocytic Leukemia.

The safety of CRISPR (clustered regularly interspaced short palindromic repeats)-based genome editing in the context of human gene therapy is largely unknown. CCR5 is a reasonable but not absolutely protective target for a cure of human immunodeficiency virus type 1 (HIV-1) infection, because CCR5-null blood cells are largely resistant to HIV-1 entry. We transplanted CRISPR-edited CCR5-ablated hematopoietic stem and progenitor cells (HSPCs) into a patient with HIV-1 infection and acute lymphoblastic leukemia. The acute lymphoblastic leukemia was in complete remission with full donor chimerism, and donor cells carrying the ablated CCR5 persisted for more than 19 months without gene editing-related adverse events. The percentage of CD4+ cells with CCR5 ablation increased by a small degree during a period of antiretroviral-therapy interruption. Although we achieved successful transplantation and long-term engraftment of CRISPR-edited HSPCs, the percentage of CCR5 disruption in lymphocytes was only approximately 5%, which indicates the need for further research into this approach. (Funded by the Beijing Municipal Science and Technology Commission and others; ClinicalTrials.gov number, NCT03164135.).

CRISPR/Cas9-Mediated CCR5 Ablation in Human Hematopoietic Stem/Progenitor Cells Confers HIV-1 Resistance In Vivo.

Transplantation of hematopoietic stem cells (HSCs) with a naturally occurring CCR5 mutation confers a loss of detectable HIV-1 in the patient, making ablation of the CCR5 gene in HSCs an ideal therapy for an HIV-1 cure. Although CCR5 disruption has been attempted in CD4+ T cells and hematopoietic stem/progenitor cells (HSPCs), efficient gene editing with high specificity and long-term therapeutic potential remains a major challenge for clinical translation. Here, we established a CRISPR/Cas9 gene editing system in human CD34+ HSPCs and achieved efficient CCR5 ablation evaluated in long-term reconstituted NOD/Prkdcscid/IL-2Rγnull mice. The CCR5 disruption efficiency in our system remained robust in secondary transplanted repopulating hematopoietic cells. More importantly, an HIV-1 resistance effect was observed as indicated by significant reduction of virus titration and enrichment of human CD4+ T cells. Hence, we successfully established a CRISPR/Cas9 mediated CCR5 ablating system in long-term HSCs, which confers HIV-1 resistance in vivo. Our study provides evidence for translating CCR5 gene-edited HSC transplantation for an HIV cure to the clinic.

Cell cycle arrest induced by inhibitors of epigenetic modifications in maize (Zea mays) seedling leaves: characterization of the process and possible mechanisms involved.

Epigenetic modifications play crucial roles in the regulation of chromatin architecture and are involved in cell cycle progression, including mitosis and meiosis. To explore the relationship between epigenetic modifications and the cell cycle, we treated maize (Zea mays) seedlings with six different epigenetic modification-related inhibitors and identified the postsynthetic phase (G2 ) arrest via flow cytometry analysis. Total H4K5ac levels were significantly increased and the distribution of H3S10ph signalling was obviously changed in mitosis under various treatments. Further statistics of the cells in different periods of mitosis confirmed that the cell cycle was arrested at preprophase. Concentrations of hydrogen peroxide were relatively higher in the treated plants and the antioxidant thiourea could negate the influence of the inhibitors. Moreover, all of the treated plants displayed negative results in the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) and γ-H2AX immunostaining assays after exposure for 3 d. Additionally, the expression level of topoisomerase genes in the treated plants was relatively lower than that in the untreated plants. These results suggest that these inhibitors of epigenetic modifications could cause preprophase arrest via reactive oxygen species formation inhibiting the expression of DNA topoisomerase genes, accompanied by changes in the H4K5ac and H3S10ph histone modifications.

Multilayered molecular profiling supported the monoclonal origin of metastatic renal cell carcinoma.

Primary renal cell carcinomas (pRCCs) have a high degree of intratumoral heterogeneity and are composed of multiple distinct subclones. However, it remains largely unknown that whether metastatic renal cell carcinomas (mRCCs) also have startling intratumoral heterogeneity or whether development of mRCCs is due to early dissemination or late diagnosis. To decipher the evolution of mRCC, we analyzed the multilayered molecular profiles of pRCC, local invasion of the vena cava (IVC), and distant metastasis to the brain (MB) from the same patient using whole-genome sequencing, whole-exome sequencing, DNA methylome profiling, and transcriptome sequencing. We found that mRCC had a lower degree of heterogeneity than pRCC and was likely to result from recent clonal expansion of a rare, advantageous subclone. Consequently, some key pathways that are targeted by clinically available drugs showed distinct expression patterns between pRCC and mRCC. From the genetic distances between different tumor subclones, we estimated that the progeny subclone giving rise to distant metastasis took over half a decade to acquire the full potential of metastasis since the birth of the subclone that evolved into IVC. Our evidence supported that mRCC was monoclonal and distant metastasis occurred late during renal cancer progression. Thus, there was a broad window for early detection of circulating tumor cells and future targeted treatments for patients with mRCCs should rely on the molecular profiles of metastases.

Experimental study on the treatment of deep miscellaneous fill foundation in Xinjiang by vibrating rod compaction method.

Currently, the treatment of miscellaneous fill foundations, composed of a mixture of domestic garbage, construction solid waste, and natural soil, presents a significant challenge in urban peripheral engineering construction. This paper discusses the application of vibrating rod compaction technology for foundation treatment in Xinjiang. It evaluates the effectiveness of cross-section vibrating rod compaction equipment in reinforcing fine-grained miscellaneous fill foundations. The study analyzes the impact of construction disturbances caused by the insertion of the vibrating rod, monitoring horizontal stresses at various depths. Both laboratory and field tests show significant improvements: soil dry density increased by 8% to 18%, porosity decreased by 10% to 23%, compression modulus increased by 22% to 246%, and compression coefficient decreased by 8% to 70%. Additionally, cohesion (C) and angle of friction (ɸ) saw increases ranging from 7 to 38% and 3% to 25%, respectively. Below a depth of 3 m, cone tip resistance exceeded 10 MPa, and sidewall friction resistance increased to over 100 kPa, surpassing pre-treatment values. The standard penetration test results doubled stroke length compared to pre-treatment, indicating a substantial improvement in foundation bearing capacity. Surface wave tests before and after treatment showed a 15% increase in wave velocity, reflecting a more compact soil structure. The vibrating rod compaction method is innovative, energy-efficient, environmentally friendly, and economically beneficial, holding great potential for future miscellaneous fill treatments.

Study on mechanical properties of interbedded rock masses with microcrack based on thermal-mechanical coupling.

The mechanical properties of deep rock masses are significantly influenced by temperature and other factors. The effect of temperature on the strength of deep rock masses will pose a serious challenge to deep resource exploitation and engineering construction. In this paper, the thermal-mechanical coupling calculation model is established by particle flow code (PFC2D) to study the uniaxial compression response of rock masses with microcracks after temperature load. The strength of failure, microcracks, and strain was analyzed. The results show that: (i) When the soft rock thickness ratio Hs/H < 0.5, the displacement caused by the applied temperature is concentrated at the structural plane, and the contact force is concentrated at the end of the initial microcrack. When Hs/H ≥ 0.5, the displacement caused by the applied temperature is concentrated on both sides of the initial microcrack, and the contact force is concentrated in the hard rock area. (ii) The number of microcracks decreases with the increase of soft rock thickness under different working conditions. When the soft rock thickness ratio Hs/H < 0.5, the relationship curve between the number of microcracks and the vertical strain shows two stages of change. When Hs/H ≥ 0.5, the relationship curve between the number of cracks and the vertical strain changes shows three stages of change. (iii) When the soft rock thickness ratio Hs/H < 0.5, the failure strength decreases with the increase of soft rock thickness ratio at T = 100°C and 200°C. When T = 300°C and 400°C, the failure strength decreased first and then increased. When Hs/H ≥ 0.5, the failure strength increases with the increase of soft rock thickness at T = 200°C, 300°C, and 400°C. At T = 100°C, the failure strength decreases with the increase of soft rock thickness.

Prediction model of land surface settlement deformation based on improved LSTM method: CEEMDAN-ICA-AM-LSTM (CIAL) prediction model.

The uneven settlement of the surrounding ground surface caused by subway construction is not only complicated but also liable to cause casualties and property damage, so a timely understanding of the ground settlement deformation in the subway excavation and its prediction in real time is of practical significance. Due to the complex nonlinear relationship between subway settlement deformation and numerous influencing factors, as well as the existence of a time lag effect and the influence of various factors in the process, the prediction performance and accuracy of traditional prediction methods can no longer meet industry demands. Therefore, this paper proposes a surface settlement deformation prediction model by combining noise reduction and attention mechanism (AM) with the long short-term memory (LSTM). The complete ensemble empirical mode decomposition with adaptive noise (CEEMDAN) and independent component analysis (ICA) methods are used to denoise the input original data and then combined with AM and LSTM for prediction to obtain the CEEMDAN-ICA-AM-LSTM (CIAL) prediction model. Taking the settlement monitoring data of the construction site of Urumqi Rail Transit Line 1 as an example for analysis reveals that the model in this paper has better effectiveness and applicability in the prediction of surface settlement deformation than multiple prediction models. The RMSE, MAE, and MAPE values of the CIAL model are 0.041, 0.033 and 0.384%; R2 is the largest; the prediction effect is the best; the prediction accuracy is the highest; and its reliability is good. The new method is effective for monitoring the safety of surface settlement deformation.

Research on the damage characteristics of rock masses based on double guide-hole blasting under high in-situ stress.

In complex high in-situ stress conditions, how to achieve the ideal rock blasting effect through effective methods is often a difficult point of blasting operations. This paper analyzes the influence of guide holes on blasting effect by adding guide holes to the rock pre-treatment method. Based on the particle expansion method to carry out double-hole blasting experiments, the influence of the blasthole spacing and ground stress on the blasting effect is investigated from the levels of macroscopic cracking effect, microscopic particle contact and so on. The study shows that: (i) the setting of empty holes between the gun holes can enhance the crack penetration effect, and the penetration effect is more obvious when the distance between the gun holes and the empty holes is less than 2.5 times the radius of the crushed zone. (ii) At the level of contact force chain, when the distance between blastholes and empty holes is less than 2.5 times the radius of the crushing zone. The compressive stress in the direction perpendicular to the direction of the blasthole line inhibits crack development, and the tensile stress in the direction parallel to the blasthole line promotes crack development. The main stress direction is perpendicular to the direction of the blasthole line. (iii) As the distance between the blastholes increases, the effect of crack suppression by stresses in the vertical direction decreases, and the main force direction is parallel to the direction of the blasthole line.

DEM modeling direct shearing behavior of sand considering anti-rotation of particle.

Considering anti-rotation of sand particles, two-dimensional Discrete Element Method (DEM) has been employed to reproduce direct shear behaviors of sand with different particle distribution sizes, so as to explore effects of anti-rotation of particle on responses of stress-displacement and dilatancy, the evolution law of shear stress, coordination number and vertical displacement of sand samples, and analyze the contact force chain, contact fabric and porosity of the samples after shearing.The results show that the anti-rotation ability of sand is enhanced, the torque of overcoming the relative rotation between particles is increased, and the peak shear stress, dilatancy and porosity in the middle of the sample are increased; with the increase of the anti-rotation coefficient, the coordination number decreases more obviously. The proportion of the contact number in the direction of 100°-160° to the total contact number decreases with the increase of the anti-rotation coefficient. The elliptical shape of the contact configuration becomes more flat, and the anisotropy of the contact force chain is more obvious; compared with fine sand, the coarse sand has greater shear capacity, more obvious dilatancy and larger porosity in the middle of the sample.The maximum minimum particle size ratio of the sample becomes larger, so that the shear strength of the sample is reduced, and the dilatancy is also weak.

Numerical simulation of size effect of defective rock under compression condition.

The existence of various types of damage, small cracks, some large voids and the size of the sample in the rock will make the experimental results show great discreteness. In this paper, based on the results of laboratory experiments, a numerical model of large flawed rock samples is established by using particle flow software PFC2D, and the mechanical response of rocks with different length-diameter ratios and different flaw positions in uniaxial compression experiments is discussed. The results show that the specimen size has a significant effect on the crack characteristics, mechanical characteristics and energy characteristics of rock mass. From the perspective of energy and crack characteristics, the total number of cracks after the failure of the defective rock sample is slightly lower than that of the intact rock sample, resulting in a slightly lower peak strain energy during the rock failure process. From the mechanical properties of rock samples, the Poisson's ratio of intact rock samples is slightly smaller than that of defective rock samples. The strength of the defective sample is weakened relative to the complete rock sample, and the relationship formula between the weakening range and the aspect ratio is obtained through analysis. Moreover, different defect locations lead to different crack processes and crack modes, resulting in different uniaxial compressive strength.

Cracks propagation characteristics of double-hole delay blasting in soft-hard composite rock mass.

By researching the distance between blasthole and interface of soft-hard rock strata, as well as the time of delay detonation, blasting effect of the rock mass will be more controllable. Firstly, validity of numerical method was authenticated from three angles: blasting coupled stress field, ratio of crushing zone radius to blasthole radius, and crack network state. Under the condition of soft-hard rock strata, numerical model of double-hole blasting was established by using PFC2D. Then delay blasting experiments were carried out under different relative positions of blasthole and interface. Ultimately, results were analyzed from three perspectives: crack network, crack quantity and rock fragment. Results show that: (1) When detonated in hard rock, if between interface and blasthole distance is greater than twice crushing zone radius, the closer blasthole is to the interface, the more obvious the "hook" phenomenon between the two blastholes is. With increasing delayed initiation time, "hook" phenomenon will weaken or even disappear. (2) Based on the crack information initiated in hard rock, the law of crack number varying with thickness of hard rock and delay time is obtained. (3) For initiation in hard rock, crack extension range is large, but less fragments are formed. The law is opposite to that initiation in structural plane and soft rock. Fragmentation area increases exponentially with increasing soft rock thickness, and exponential function is obtained.

Study of snow cover/depth evolution characteristics in Tianshan region of China based on geographical partition.

Based on the digital elevation data, snow depth and snow cover remote sensing data, this paper divides six snow evolution areas and geographical partitions, extracts the geographical partitions of each evolution area and obtains the geographical characteristics of the evolution area for analysis. The results show that: (1) From 2003 to 2017, the average snow area decreased at a rate of - 0.004, and the average snow depth increased at a rate of 0.03. (2) The snow in the middle altitude hill with shady gentle slope area is the most obvious in the seasonal evolution, and the percentage of this region in the seasonal snow evolution area is 5.46%, the snow depth in the middle altitude hill with sunny and gentle slopes area increased and decreased significantly in the past 15 years, and the percentage of this region in the SD significant changes evolution area was 6.32%. The snow in the low relief middle altitude mountain with shady and moderate slope area not only shows obvious seasonal evolution, but also increases and decreases significantly in snow depth. And the percentage of this region in the seasonal snow significant evolution area is 5.82%. (3) The geographical partitions with the largest area in all evolution areas is the middle altitude hill with sunny and gentle slopes area (4.75%). (4) The geographical partition with the largest variation of snow depth in Tianshan region is the low relief middle altitude mountain with shady and moderate slope area (12.02 cm). (5) The snow accumulation and melting are obvious in the range of 1000-3500 m above altitude, different geomorphology types lead to obvious differences in snow characteristics. The snow melting is most obvious in the gentle slope area of the low topographic relief geomorphology types, and the snow accumulation is most obvious in the steep slope area of the middle relief geomorphology types.

Influence of external insert angle on structural deformation of long pipe shed with shallow hole.

The long pipe shed advanced support has the characteristics of large construction difficulty, wide support range, and easy deviation. The difference in construction dip angle will produce a different plastic zone of surrounding rock and supporting effects, and the rationality of advanced support design plays an important role in the safety of tunnel opening excavation. Based on the Tianshan Tunnel project, this paper aims at the problems of the loose pebble layer structure, poor cohesion, strong permeability, shallow excavation is not easy to form a confined arch, and easy to causes surrounding rock deformation and failure. Combined with the new method theory, the finite difference software FLAC3D was used to simulate seven excavation schemes of the shallow tunnel entrances. The mechanism and effect of advance support and the influence of construction angle on support effect are analyzed, and the simulation values are compared with the measured data. The results show that using long pipe shed advanced support can effectively reduce the disturbance of excavation to lose pebble rock mass and reduce the convergence value of surrounding rock. The plastic zone of surrounding rock produced by different angles can be divided into three parts. The structural stability difference of the C1 zone is small, and that of the C3 zone is large. The bending moment, shear force, and shape variables of the pipe shed decrease with the increase of the dip angle, while the axial force increases with the increase of the dip angle. According to the similarity between the simulated curve and the measured curve, the best supporting effect can be achieved when the dip angle of the pre-supporting pipe shed is set in the range of 0° ~ 3°.

Study on blasting characteristics of rock mass with weak interlayer based on energy field.

In order to explore the influence of weak interlayer on blasting characteristics in natural rock mass, by using the particle flow code (PFC2D), a single hole blasting numerical model of hard rock with soft interlayer is established. The blasting experiments at different positions and thicknesses of weak interlayer are carried out. Then an in-depth analysis from the perspectives of crack effect, stress field and energy field is made. Results showed that: (i) When the explosion is initiated outside the weak interlayer, if the interlayer is located within about twice the radius of the crushing area, the closer the interlayer is to the blast hole, the higher the damage degree of the rock mass around the blast hole. And the number of cracks will increase by about 1-2% when the distance between the weak interlayer and the blast hole decreases by 0.5 m. (ii) When detonating outside the weak interlayer, if the interlayer is within about 4 times radius of the crushing area, the hard rock on both sides of the weak interlayer will in a high stress state. Under the same case, the peak kinetic energy and peak friction energy will increase by about 23 and 13%, respectively, and the peak strain energy will increase by about 218 kJ for every 0.1 m increase in the thickness of the weak interlayer.

Effects of prophylactic antibiotic administration and antibiotic timing on culture results and clinical outcomes of paediatric musculoskeletal infection: a protocol for a randomised controlled clinical trial.

INTRODUCTION: Musculoskeletal infection (MSI) is a common cause of morbidity among the paediatric population. Some clinicians recommend withholding prophylactic antibiotics until culture collection with an aim to improve the culture sensitivity. However, a recent retrospective study reported that prophylactic antibiotic administration did not affect culture sensitivities in either disseminated or local MSI in paediatric population, which is surprising. The aim of the present study is to investigate the effects of prophylactic antibiotic administration and the timing of antibiotic administration on culture sensitivity and clinical outcomes of paediatric MSI. METHODS AND ANALYSIS: A randomised controlled clinical trial will be carried out. Individuals aged 0-18 years with a diagnosis of MSI will be screened and evaluated at the Shenzhen Children's Hospital. The participants will be randomly allocated into four groups, and they will receive the antibiotic treatment at different time points, that is, 1 week, 3 days, 1 day prior to tissue culture collection and 1 day after tissue culture collection, respectively. The primary outcome will be culture sensitivity. In addition, the disease-related markers including white blood cell count, C reactive protein, erythrocyte sedimentation rate, vital signs as well as the length of hospital stay will be measured or recorded accordingly. Using χ2 tests, the rates of positive cultures will be compared between different groups. Statistical comparisons between the different patient groups regarding the confounding and outcome variables will be conducted using independent t-tests, Mann-Whitney U tests, χ2 tests and Fisher's exact tests as appropriate with the significance level set to 5% (p<0.05). ETHICS AND DISSEMINATION: This study has received ethical approval. The findings will be disseminated both in scientific conferences and peer-reviewed journal. TRIAL REGISTRATION NUMBER: ChiCTR2100041631.

Efficient derivation of extended pluripotent stem cells from NOD-scid Il2rg-/- mice.

Recently we have established a new culture condition enabling the derivation of extended pluripotent stem (EPS) cells, which, compared to conventional pluripotent stem cells, possess superior developmental potential and germline competence. However, it remains unclear whether this condition permits derivation of EPS cells from mouse strains that are refractory or non-permissive to pluripotent cell establishment. Here, we show that EPS cells can be robustly generated from non-permissive NOD-scid Il2rg-/- mice through de novo derivation from blastocysts. Furthermore, these cells can also be efficiently generated by chemical reprogramming from embryonic NOD-scid Il2rg-/- fibroblasts. NOD-scid Il2rg-/- EPS cells can be expanded for more than 20 passages with genomic stability and can be genetically modified through gene targeting. Notably, these cells contribute to both embryonic and extraembryonic lineages in vivo. More importantly, they can produce chimeras and integrate into the E13.5 genital ridge. Our study demonstrates the feasibility of generating EPS cells from refractory mouse strains, which could potentially be a general strategy for deriving mouse pluripotent cells. The generation of NOD-scid Il2rg-/- EPS cell lines permits sophisticated genetic modification in NOD-scid Il2rg-/- mice, which may greatly advance the optimization of humanized mouse models for biomedical applications.

Rapid generation of gene-targeted EPS-derived mouse models through tetraploid complementation.

One major strategy to generate genetically modified mouse models is gene targeting in mouse embryonic stem (ES) cells, which is used to produce gene-targeted mice for wide applications in biomedicine. However, a major bottleneck in this approach is that the robustness of germline transmission of gene-targeted ES cells can be significantly reduced by their genetic and epigenetic instability after long-term culturing, which impairs the efficiency and robustness of mouse model generation. Recently, we have established a new type of pluripotent cells termed extended pluripotent stem (EPS) cells, which have superior developmental potency and robust germline competence compared to conventional mouse ES cells. In this study, we demonstrate that mouse EPS cells well maintain developmental potency and genetic stability after long-term passage. Based on gene targeting in mouse EPS cells, we established a new approach to directly and rapidly generate gene-targeted mouse models through tetraploid complementation, which could be accomplished in approximately 2 months. Importantly, using this approach, we successfully constructed mouse models in which the human interleukin 3 (IL3) or interleukin 6 (IL6) gene was knocked into its corresponding locus in the mouse genome. Our study demonstrates the feasibility of using mouse EPS cells to rapidly generate mouse models by gene targeting, which have great application potential in biomedical research.

Correction to: Efficient derivation of extended pluripotent stem cells from NOD-scid Il2rg-/- mice.

In the original publication Fig. 1D and supplementary material is incorrect. The correct figure and supplementary material is provided in this correction.

Whole-genome and whole-exome sequencing of bladder cancer identifies frequent alterations in genes involved in sister chromatid cohesion and segregation.

Bladder cancer is one of the most common cancers worldwide, with transitional cell carcinoma (TCC) being the predominant form. Here we report a genomic analysis of TCC by both whole-genome and whole-exome sequencing of 99 individuals with TCC. Beyond confirming recurrent mutations in genes previously identified as being mutated in TCC, we identified additional altered genes and pathways that were implicated in TCC. Notably, we discovered frequent alterations in STAG2 and ESPL1, two genes involved in the sister chromatid cohesion and segregation (SCCS) process. Furthermore, we also detected a recurrent fusion involving FGFR3 and TACC3, another component of SCCS, by transcriptome sequencing of 42 DNA-sequenced tumors. Overall, 32 of the 99 tumors (32%) harbored genetic alterations in the SCCS process. Our analysis provides evidence that genetic alterations affecting the SCCS process may be involved in bladder tumorigenesis and identifies a new therapeutic possibility for bladder cancer.