A novel whole-cell lysate kinase assay identifies substrates of the p38 MAPK in differentiating myoblasts

<p>Abstract</p> <p>Background</p> <p>The p38α mitogen-activated protein kinase (MAPK) is a critical mediator of myoblast differentiation, and does so in part through the phosphorylation and regulation of several transcription factors and chromatin remodelling proteins. However, whether p38α is involved in processes other than gene regulation during myogenesis is currently unknown, and why other p38 isoforms cannot compensate for its loss is unclear.</p> <p>Methods</p> <p>To further characterise the involvement of p38α during myoblast differentiation, we developed and applied a simple technique for identifying relevant <it>in vivo </it>kinase substrates and their phosphorylation sites. In addition to identifying substrates for one kinase, the technique can be used <it>in vitro </it>to compare multiple kinases in the same experiment, and we made use of this to study the substrate specificities of the p38α and β isoforms.</p> <p>Results</p> <p>Applying the technique to p38α resulted in the identification of seven <it>in vivo </it>phosphorylation sites on six proteins, four of which are cytoplasmic, in lysate derived from differentiating myoblasts. An <it>in vitro </it>comparison with p38β revealed that substrate specificity does not discriminate these two isoforms, but rather that their distinguishing characteristic appears to be cellular localisation.</p> <p>Conclusion</p> <p>Our results suggest p38α has a novel cytoplasmic role during myogenesis and that its unique cellular localisation may be why p38β and other isoforms cannot compensate for its absence. The substrate-finding approach presented here also provides a necessary tool for studying the hundreds of protein kinases that exist and for uncovering the deeper mechanisms of phosphorylation-dependent cell signalling.</p

Progress in targeted observation for meso-scale convective system and some thoughts on its applications to convection nowcasting in large cities

Improving the forecasting skills of the meso-scale convective system (MCS) is one of the key scientific problems in the field of numerical weather prediction. The occurrence and development of severe convective weather are affected by multiple factors such as atmospheric thermodynamic and kinetic conditions, topography, and air pollution conditions. Due largely to the uncertainty in models and the inevitable errors of initial values, large uncertainties still exist for the accurate prediction of severe weather produced by the MCS. Therefore, to effectively improve the accuracy of severe convective weather forecasts in China, conducting targeted observation experiments in key areas of interest, which typically helps to reduce the uncertainty level of the model's initial meteorological field, may be one of the effective ways forward. It follows that the initiation and formation mechanisms of MCS can be revealed, and the forecast skills of severe convection will be improved. In this paper, we first propose the technology roadmap of targeted observation as follows. Based on the modern meteorological observational network over the Jing-Jin-Ji area, the typical MCS forecast sensitive regions are identified by using the Ensemble Transform Kalman filter, combined with the atmospheric sounding systems mounted on the mobile vehicle, where targeted observation experiments will be conducted. As such, convective initiation mechanisms are elucidated, and the novel methods expected to improve the forecast skill for MCS are explored. Secondly, in response to the challenge of "finding needles in a haystack" in the vertical observation of the lower atmosphere for short-term forecasting and warning of severe convective weather in large cities, the potential application value of a dynamic triangular observation mesonet in the study of triggering and development mechanisms of severe convective weather was explored through the construction of a Pyramid-shaped LOwer Tropospheric Observational System (PLOTOS) that consists of five stations with simultaneous vertical observation capabilities. Finally, it is suggested that the initiation and formation mechanisms of severe convective weather be unraveled using PLOTOS, and the mesoscale targeted observation technology be developed, which is crucial to the improvement of weather observational networks in large cities and provide new ideas and methods for improving the forecast of severe convective weather processes

Targeting LIF-mediated paracrine interaction for pancreatic cancer therapy and monitoring.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis largely owing to inefficient diagnosis and tenacious drug resistance. Activation of pancreatic stellate cells (PSCs) and consequent development of dense stroma are prominent features accounting for this aggressive biology1,2. The reciprocal interplay between PSCs and pancreatic cancer cells (PCCs) not only enhances tumour progression and metastasis but also sustains their own activation, facilitating a vicious cycle to exacerbate tumorigenesis and drug resistance3-7. Furthermore, PSC activation occurs very early during PDAC tumorigenesis8-10, and activated PSCs comprise a substantial fraction of the tumour mass, providing a rich source of readily detectable factors. Therefore, we hypothesized that the communication between PSCs and PCCs could be an exploitable target to develop effective strategies for PDAC therapy and diagnosis. Here, starting with a systematic proteomic investigation of secreted disease mediators and underlying molecular mechanisms, we reveal that leukaemia inhibitory factor (LIF) is a key paracrine factor from activated PSCs acting on cancer cells. Both pharmacologic LIF blockade and genetic Lifr deletion markedly slow tumour progression and augment the efficacy of chemotherapy to prolong survival of PDAC mouse models, mainly by modulating cancer cell differentiation and epithelial-mesenchymal transition status. Moreover, in both mouse models and human PDAC, aberrant production of LIF in the pancreas is restricted to pathological conditions and correlates with PDAC pathogenesis, and changes in the levels of circulating LIF correlate well with tumour response to therapy. Collectively, these findings reveal a function of LIF in PDAC tumorigenesis, and suggest its translational potential as an attractive therapeutic target and circulating marker. Our studies underscore how a better understanding of cell-cell communication within the tumour microenvironment can suggest novel strategies for cancer therapy

Curing hemophilia A by NHEJ-mediated ectopic F8 insertion in the mouse

BACKGROUND: Hemophilia A, a bleeding disorder resulting from F8 mutations, can only be cured by gene therapy. A promising strategy is CRISPR-Cas9-mediated precise insertion of F8 in hepatocytes at highly expressed gene loci, such as albumin (Alb). Unfortunately, the precise in vivo integration efficiency of a long insert is very low (~ 0.1%). RESULTS: We report that the use of a double-cut donor leads to a 10- to 20-fold increase in liver editing efficiency, thereby completely reconstituting serum F8 activity in a mouse model of hemophilia A after hydrodynamic injection of Cas9-sgAlb and B domain-deleted (BDD) F8 donor plasmids. We find that the integration of a double-cut donor at the Alb locus in mouse liver is mainly through non-homologous end joining (NHEJ)-mediated knock-in. We then target BDDF8 to multiple sites on introns 11 and 13 and find that NHEJ-mediated insertion of BDDF8 restores hemostasis. Finally, using 3 AAV8 vectors to deliver genome editing components, including Cas9, sgRNA, and BDDF8 donor, we observe the same therapeutic effects. A follow-up of 100 mice over 1 year shows no adverse effects. CONCLUSIONS: These findings lay the foundation for curing hemophilia A by NHEJ knock-in of BDDF8 at Alb introns after AAV-mediated delivery of editing components