Generation of Inducible CRISPRi and CRISPRa Human Stromal/Stem Cell Lines for Controlled Target Gene Transcription during Lineage Differentiation

Background. Human bone marrow stromal/stem cells (hMSCs, also known as the skeletal stem cells or mesenchymal stem cells) are being employed to study lineage fate determination to osteoblasts, adipocytes, and chondrocytes. However, mechanistic studies employing hMSC have been hampered by the difficulty of deriving genetically modified cell lines due to the low and unstable transfection efficiency. Methods. We infected hMSC with a CRISPR/Cas9 lentivirus system, with specific inducible dCas9-coupled transcription activator or repressor: dCas9-KRAB or dCas9-VP64, respectively, and established two hMSC lines (hMSC-CRISPRi and hMSC-CRISPRa) that can inhibit or activate gene expression, respectively. The two cell lines showed similar cell morphology, cell growth kinetics, and similar lineage differentiation potentials as the parental hMSC line. The expression of KRAB-dCas9 or VP64-dCas9 was controlled by the presence or absence of doxycycline (Dox) in the cell culturing medium. To demonstrate the functionality of the dCas9-effector hMSC system, we tested controlled expression of alkaline phosphatase (ALP) gene through transfection with the same single ALP sgRNA. Results. In the presence of Dox, the expression of ALP showed 60-90% inhibition in hMSC-CRISPRi while ALP showed more than 20-fold increased expression in hMSC-CRISPRa. As expected, the ALP was functionally active and the cells showed evidence for inhibition or enhancement of in vitro osteoblast differentiation, respectively. Conclusion. hMSC-CRISPRi and hMSC-CRISPRa are useful resources to study genes and genetic pathways regulating lineage-specific differentiation of hMSC

Actin depolymerization enhances adipogenic differentiation in human stromal stem cells

Human stromal stem cells (hMSCs) differentiate into adipocytes that play a role in skeletal tissue homeostasis and whole body energy metabolism. During adipocyte differentiation, hMSCs exhibit significant changes in cell morphology suggesting changes in cytoskeletal organization. Here, we examined the effect of direct modulation of actin microfilament dynamics on adipocyte differentiation. Stabilizing actin filaments in hMSCs by siRNA-mediated knock down of the two main actin depolymerizing factors (ADFs): Cofilin 1 (CFL1) and Destrin (DSTN) or treating the cells by Phalloidin reduced adipocyte differentiation as evidenced by decreased number of mature adipocytes and decreased adipocyte specific gene expression (ADIPOQ, LPL, PPARG, FABP4). In contrast, disruption of actin cytoskeleton by Cytochalasin D enhanced adipocyte differentiation. Follow up studies revealed that the effects of CFL1 on adipocyte differentiation depended on the activity of LIM domain kinase 1 (LIMK1) which is the major upstream kinase of CFL1. Inhibiting LIMK by its specific chemical inhibitor LIMKi inhibited the phosphorylation of CFL1 and actin polymerization, and enhanced the adipocyte differentiation. Moreover, treating hMSCs by Cytochalasin D inhibited ERK and Smad2 signaling and this was associated with enhanced adipocyte differentiation. On the other hand, Phalloidin enhanced ERK and Smad2 signaling, but inhibited adipocyte differentiation which was rescued by ERK specific chemical inhibitor U0126. Our data provide a link between restructuring of hMSCs cytoskeleton and hMSCs lineage commitment and differentiation. Keywords: Actin cytoskeleton, Actin depolymerizing factors, Adipocyte differentiation, Human stromal stem cell

Perspectives on Gamma-Ray Burst Physics and Cosmology with Next Generation Facilities

High-redshift Gamma-Ray Bursts (GRBs) beyond redshift $\sim6$ are potentially powerful tools to probe the distant early Universe. Their detections in large numbers and at truly high redshifts call for the next generation of high-energy wide-field instruments with unprecedented sensitivity at least one order of magnitude higher than the ones currently in orbit. On the other hand, follow-up observations of the afterglows of high-redshift GRBs and identification of their host galaxies, which would be difficult for the currently operating telescopes, require new, extremely large facilities of at multi-wavelengths. This chapter describes future experiments that are expected to advance this exciting field, both being currently built and being proposed. The legacy of Swift will be continued by SVOM, which is equipped with a set of space-based multi-wavelength instruments as well as and a ground segment including a wide angle camera and two follow-up telescopes. The established Lobster-eye X-ray focusing optics provides a promising technology for the detection of faint GRBs at very large distances, based on which the {THESEUS}, {Einstein Probe} and other mission concepts have been proposed. Follow-up observations and exploration of the reionization era will be enabled by large facilities such as {SKA} in the radio, the 30m class telescopes in the optical/near-IR, and the space-borne {WFIRST} and {JWST} in the optical/near-IR/mid-IR. In addition, the X-ray and $\gamma$-ray polarization experiment POLAR is also introduced.Comment: accepted for publication in Space Science Review; reprinted as a chapter in a book of the Space Sciences Series of ISSI for the proceedings of the ISSI-Beijing workshop " Gamma-Ray Bursts: a Tool to Explore the Young Universe

Einstein Probe - a small mission to monitor and explore the dynamic X-ray Universe

Einstein Probe is a small mission dedicated to time-domain high-energy astrophysics. Its primary goals are to discover high-energy transients and to monitor variable objects in the $0.5-4~$keV X-rays, at higher sensitivity by one order of magnitude than those of the ones currently in orbit. Its wide-field imaging capability, featuring a large instantaneous field-of-view ($60^\circ \times60^\circ$, $\sim1.1$sr), is achieved by using established technology of micro-pore (MPO) lobster-eye optics, thereby offering unprecedentedly high sensitivity and large Grasp. To complement this powerful monitoring ability, it also carries a narrow-field, sensitive follow-up X-ray telescope based on the same MPO technology to perform follow-up observations of newly-discovered transients. Public transient alerts will be downlinked rapidly, so as to trigger multi-wavelength follow-up observations from the world-wide community. Over three of its 97-minute orbits almost the entire night sky will be sampled, with cadences ranging from 5 to 25 times per day. The scientific objectives of the mission are: to discover otherwise quiescent black holes over all astrophysical mass scales by detecting their rare X-ray transient flares, particularly tidal disruption of stars by massive black holes at galactic centers; to detect and precisely locate the electromagnetic sources of gravitational-wave transients; to carry out systematic surveys of X-ray transients and characterize the variability of X-ray sources. Einstein Probe has been selected as a candidate mission of priority (no further selection needed) in the Space Science Programme of the Chinese Academy of Sciences, aiming for launch around 2020.Comment: accepted to publish in PoS, Proceedings of "Swift: 10 Years of Discovery" (Proceedings of Science; ed. by P. Caraveo, P. D'Avanzo, N. Gehrels and G. Tagliaferri). Minor changes in text, references update

Experimental investigation about the influence of airflow on droplet sizes of mechanical nozzles for coal mining face

Visokotlačno raspršivanje je jedna od primarnih metoda za sprječavanje prašine u potpuno mehaniziranom iskopu, a učinci raspršivanja mehaničkih mlaznica značajno utječu na učinak raspršivanja. Do sada su mlaznice uglavnom odabirane subjektivnom proizvoljnošću tehničara, utječući na poboljšanje učinka suzbijanja prašine. Iz tog razloga, prije i poslije simulacije stvarne brzine zraka iskopa ugljena, provedeni su usporedni pokusi učinaka atomizacije na šest vrsta najčešće korištenih mehaničkih mlaznica s vlastitom platformom za prskanje. Na osnovu podataka o ispitivanju dobivene su prosječne veličine kapljica i redoslijed kvalitete raspršivanja različitih mehaničkih mlaznica, a osim toga je induciran utjecaj turbulencije protoka zraka na veličine kapljica. Konačno, prema eksperimentalnim rezultatima, mlaznice koje se koriste u vanjskom raspršivaču su odabrane za #10310 radnih iskopa rudnika ugljena Xinglongzhuang u Kini, koji je postigao velike učinke smanjenja prašine.High-pressure spraying is one of the primary methods for dust prevention in coal mine fully mechanized caving face, and the atomization effects of mechanical nozzles influence dedusting result significantly. While so far, the nozzles are selected by technician’s subjective arbitrariness mainly, affecting improving dedusting effect. For this reason, before and after simulating actual air velocity of coal workface, the comparative experiments on the atomization effects of the six kinds of commonly used mechanical nozzles were conducted with self-designed spraying platform. Based on the test data, the average droplets’ size and the atomization quality order of different mechanical nozzles were obtained, moreover, the airflow’s turbulence influence on droplets’ sizes was induced. Finally, according to the experimental results, nozzles used in external spray of shearer were selected for #10310 working face of Xinglongzhuang coal mine in China, which had achieved great dust reduction effects

Whole Genome Sequencing and Comparative Genomics Analyses of Pandoraea sp. XY-2, a New Species Capable of Biodegrade Tetracycline

Few bacteria are resistant to tetracycline and can even biodegrade tetracycline in the environment. In this study, we isolated a bacterium Pandoraea sp. XY-2, which could biodegrade 74% tetracycline at pH 7.0 and 30°C within 6 days. Thereafter, we determined the whole genome sequence of Pandoraea sp. XY-2 genome is a single circular chromosome of 5.06 Mb in size. Genomic annotation showed that two AA6 family members-encoding genes and nine glutathione S-transferase (GSTs)-encoding genes could be relevant to tetracycline biodegradation. In addition, the average nucleotide identities (ANI) analysis between the genomes of Pandoraea sp. XY-2 and other Pandoraea spp. revealed that Pandoraea sp. XY-2 belongs to a new species. Moreover, comparative genome analysis of 36 Pandoraea strains identified the pan and specific genes, numerous single nucleotide polymorphisms (SNPs), insertions, and deletion variations (InDels) and different syntenial relationships in the genome of Pandoraea sp. XY-2. Finally, the evolution and the origin analysis of genes related to tetracycline resistance revealed that the six tetA(48) genes and two specificgenes tetG and tetR in Pandoraea sp. XY-2 were acquired by horizontal gene transfer (HGT) events from sources related to Paraburkholderia, Burkholderia, Caballeronia, Salmonella, Vibrio, Proteobacteria, Pseudomonas, Acinetobacter, Flavimaricola, and some unidentified sources. As a new species, Pandoraea sp. XY-2 will be an excellent resource for the bioremediation of tetracycline-contaminated environment

Comparative proteomic analysis reveals alterations in development and photosynthesis-related proteins in diploid and triploid rice

Six pairs of primers were designed for gene-specific transcript amplification. (DOC 25 kb

A Humanized Anti-VEGF Rabbit Monoclonal Antibody Inhibits Angiogenesis and Blocks Tumor Growth in Xenograft Models

Rabbit antibodies have been widely used in research and diagnostics due to their high antigen specificity and affinity. Though these properties are also highly desirable for therapeutic applications, rabbit antibodies have remained untapped for human disease therapy. To evaluate the therapeutic potential of rabbit monoclonal antibodies (RabMAbs), we generated a panel of neutralizing RabMAbs against human vascular endothelial growth factor-A (VEGF). These neutralizing RabMAbs are specific to VEGF and do not cross-react to other members of the VEGF protein family. Guided by sequence and lineage analysis of a panel of neutralizing RabMAbs, we humanized the lead candidate by substituting non-critical residues with human residues within both the frameworks and the CDR regions. We showed that the humanized RabMAb retained its parental biological properties and showed potent inhibition of the growth of H460 lung carcinoma and A673 rhabdomyosarcoma xenografts in mice. These studies provide proof of principle for the feasibility of developing humanized RabMAbs as therapeutics