Abstract:Cell-free protein synthesis is an in-vitro cell-free expression method for protein synthesis. High-throughput protein expression and in-vitro reconstitution of membrane proteins can be realized in CFPS-containing artificial cells. This review describes various CFPS systems including E. coli extracts, rabbit reticulocytes extracts, wheat germ extracts, and yeast extracts, and summarizes the progress of cell free protein synthesis inside artificial cells. The challenges and the future directions of this field are also proposed at the end of this paper.

Abstract:Exosomes are extracellular vesicles (30-150nm) that are secreted into a various of bodily fluids and interstitial space by all types of cells. Circular RNAs (circRNAs) are a class of non-coding RNA (ncRNA) generated from the precursor mRNA (pre-mRNA) backsplicing process. Recent evidence suggests that circRNAs enriched in exosome, and play an important role in tumor microenvironment. In this review, we discuss our claim that the biogenesis of exosomal circRNAs, including the regulation mechanism of circRNAs sorting into exosomes and releasing of exosomes. Then, we elucidated the role and mechanism of exosomal circRNAs regulate EMT, cytoskeleton, angiogenesis, metabolism and inflammation, immunity and drug resistance in the tumor microenvironment. Finally, we discussed the clinical application prospects and value of exosomal circRNAs as tumor markers and therapeutic targets.

Abstract:Malignant tumors are seriously harmful to human health. At present, there are mainly three ways to treat them: surgery, radiotherapy and chemotherapy. However, the curative effect is not satisfactory. So it is very urgent to find a new target for tumor treatment and achieve targeted treatment. Warburg effect is common in many kinds of tumors. Its important feature is that under the condition of sufficient oxygen, the energy metabolism of cancer cells is still dominated by glycolysis. Warburg effect is a typical process of glycolysis in which glucose is absorbed in large quantities and converted into lactic acid by glycolysis. Lactate, a glycolytic product, can activate many important signaling pathways in cancer cells, and promote the survival, invasion, immune escape, metastasis and angiogenesis of cancer cells. Therefore, targeting lactate metabolism and its key enzymes may provide new targets for tumor therapy. This paper reviews the metabolic process of lactate, the change of lactate metabolism mode of tumor cells, the immune escape of lactate to tumor cells, tumor metastasis, tumor angiogenesis, and the treatment of tumor with lactate as the target.

Abstract:Periaqueductal gray(PAG) plays an important role in pain modulation. It is not only an important participant of ascending pain transmission pathways, but also a critical part of descending pain modulation pathways. In PAG, there are evidences that several neurotransmitters (such as γ-aminobutyric acid, 5-hydroxytryptamine, glutamate) and neuromodulators (such as endogenous opioid peptide, cannabinoid) are involved in the information transmission and functional regulation of pain. PAG not only regulates pain locally through their own level changes and interaction of these factors, but also regulates pain by receiving fiber projections from other brain regions and sending fiber projections to other pain related brain regions. For example, the analgesic effect of the increased level of GABA in PAG could be realized by the effect of opioid peptides on GABAergic interneurons, or driven directly by the fiber projection from cortex to the GABAergic interneurons in PAG. GABAergic neurons in PAG can also regulate pain by affecting the NAergic fibers projected from A7 cells to the spinal cord. Thus, PAG participates pain modulation in complicated and multiple ways.

Abstract:Transient receptor potential (TRP) channels are widely distributed in nervous and non-nervous systems, responding to thermal, chemical, mechanical and other stimuli. They are emerging as essential sensory molecules that allow animals to respond to environmental changes. The 27 members of the mammalian TRP superfamily are grouped into six subfamilies, the TRPA (ankyrin) family, the TRPC (canonical) family, the TRPM (melastatin) family, the TRPML (mucolipin) family, the TRPP (polycystin) family, and the TRPV (vanilloid) family on the basis of amino acid sequence homology. The TRPV subfamily is composed of six members, TRPV1-6. They can be further divided into two groups, TRPV1-4 subgroup which is weakly Ca²⁺-selective and highly sensitive to heat, and TRPV5, TRPV6 subgroup which is highly Ca²⁺-selective and not heat-sensitive. Mounting evidences suggest that TRPV channels are responsible for membrane potential regulation and intracellular Ca²⁺ signaling, and thus regulate a large number of cellular functions, such as temperature sensation and vasodilation. Dysfunction in channel expression and/or activity has been linked to human disease like cancer and cardiovascular disease. Post-translational modifications (PTMs) involving a functional group being added to a protein, a chemical change in amino acids, and a structural change in the protein have been implicated in regulating activity, localization and interaction with other cellular molecules, thereby increasing the functional diversity of the proteome. Many studies show that TRPV subfamily channels can also undergo post-translational modifications and PTMs have an important impact on channel function. The aim of this review is to address the major advances regarding the various post-translational modifications such as phosphorylation, glycosylation, ubiquitination, SUMOylation, and covalent modification that have been reported to regulate the functions of TRPV channels. Furthermore, we also discussed the possible functions of TRPV channels before and after PTMs in physiological or pathological activities, for a better understanding of the relationship between PTM and physiological or pathological activities.

Abstract:PDZ-binding kinase (PBK), a member of the mitogen-activated protein kinase kinase (MAPKK) family, is a silk-threonine kinase containing 322 amino acids. Its protein is mainly expressed in the tissues with high proliferation potential, testis, placenta, activated T cells and neural progenitor cells while extremely low in the tissues and cells with a high degree of differentiation. In recent years, PBK expression has been found significantly enhanced in a variety of malignant tumor such as breast, colon, liver, lung, prostate, esophageal cancer, and so on, closely related to poor prognosis of the cancers mentioned above. Further studies have pointed out that PBK can also promote the proliferation, invasion and metastasis of many cancer cells through a series of complex signaling pathways including Wnt, PI3K/AKT/mTOR, MAPK, FOXM1, nuclear factor-κB and matrix metalloproteinase (MMP), and participate in drug resistance. Moreover, the role of PBK has been confirmed controlled by different microRNA like miR-216b-3p, miR-770-5p and miR-372-5p in the cancers. All above suggest that PBK might be a new proto-oncogene, which is expected to become a new molecular target for tumor suppressor drugs.

Abstract:Ash2l promotes H3K4 trimethylation levels through methyltransferase MLL/SET1 complex, which is essential for mouse embryonic development. Ash2l have two isoforms (Ash2l-1 and Ash2l-2) through alternative promoter usage in mouse. However, the mechanism of this gene in mouse embryonic development and the function of different isoforms remain unknown. In this study, we used CRISPR/Cas9 technology to specifically disrupt Ash2l-1 in mice and investigated the role of Ash2l-1 in mouse early embryonic development. Disruption of Ash2l-1 resulted in embryonic lethality at E9.5-10.5. Particularly, E9.5 Ash2l-1-deficient embryos exhibited severe growth defects, including developmental defects of yolk sac. Gene expression profiling showed that Ash2l-1 deficiency affected the expression of specific genes involved in erythropoiesis and vascular formation. CUT&RUN analysis showed that H3K4me3 levels of the promoter of some specific genes involved in erythropoiesis and vascular formation was down-regulated. Taken together, these results indicate that Ash2l-1 regulates gene expression through H3K4me3 and plays an essential role in the early hematopoiesis of mouse yolk sacs.

Abstract:Tumor has greatly threatened to human life. Now the main means of tumor treatment are surgery and(/or) chemoradiotherapy. However, the chemoradiotherapy is usually not suitable to sustained treatment of tumors due to its poor cell selectivity, side effects and inducing tumor cells resistance. Therefore, it is urgent to develop new drugs with low toxicity and high activity at the tumor sites. In situ self-assembly peptides which can target the tumors and self-assemble into specific nanostructures induced by the specific and highly expressed substances at the tumor tissues are expected to be a new kind of anti-tumor drugs. In this study, we designed a peptide denoted as Fmoc-FFRIKFERQ-OH based on the concept of in situ self-assembly of peptides. This peptide can target lysosomes and self-assemble in situ after being degraded by cathepsin L in lysosomes. The self-assembly properties of Fmoc-FFRIKFERQ-OH and its enzymolysis products degraded by cathepsin L were oberved by AFM and MALDI-TOF-MS. The anti-tumor activity of this peptide was measured by using the MTT and flow cytometry assay. The results demonstrated that cathepsin L could precisely hydrolyze the Fmoc-FFRIKFERQ-OH molecule under acidic conditions in vitro, and its enzymolysis product Fmoc-FFR-OH self-assembled into long nanofibers, which had high antitumor activity against both A375 and SH-SY5Y cells. The relative survival rate was only 36.08% and 25.56% for A375 and SH-SY5Y, respectively. Comparatively, Fmoc-FFRIKFERQ-OH had lower toxic and side effects on normal cells L929. The self-assembled long fibers formed by Fmoc-FFR-OH can disrupt the membrane of lysosome, which can induce cell apoptosis and(/or) death. Thus, the designed Fmoc-FFRIKFERQ-OH is expected to be a new anti-tumor drug due to its high antitumor activity and low toxicity against to normal cells.

Abstract:Data-dependent acquisition (DDA) and data-independent acquisition (DIA) based label-free quantification (LFQ) and tandem mass tag (TMT) based isotope labeling quantification are two key techniques for quantitative proteomics. Here we optimized the latest Orbitrap Exploris 480 mass spectrometer parameters in DDA, FAIMS DDA, FAIMS DIA based LFQ and TMT, and show its performance in cell line proteomics, single-cell proteomics, plasma proteomics and yeast proteomics. The results showed that the collision energy of 27, the resolution of the fragment spectrum of 15K, and maximum ion injection time of 22 ms are the best parameter combination for DDA experiment. For the proteomic analysis of ultra-low samples ranging from 200 pg-5 ng, the individual mass spectrometer parameters should be considered. We identified 1 259 and 1 725 proteins in 200 pg and 500 pg of HeLa cell lysate, respectively, achieving deep coverage of single-cell proteomics. In FAIMS DDA experiment, we chose CV-45V for 60 min or 90 min gradient, CV-45V-65V combinations for 120 min or 150 min gradient to obtain the optimal protein identifications, and identified 6 300, 6 994 and 7 500 proteins in 60 min, 120 min and 150 min from 293T proteome, respectively. In FAIMS DIA experiment, we used CV-45V-65V voltage sweeping, 60 isolation windows, and obtained the optimal proteins identifications and quantitative reproducibility. We quantified 7 019 proteins in 293T cell lysate and 1 077 proteins in depleted plasma in 60 min gradient. The combination of APD on, "Precursor Fit" threshold of 70 and Turbo TMT could simultaneously increase the number of protein identifications and the accuracy of TMT quantification. 10 989 peptides and 2 162 protein were quantified in TMT11-plex labeled yeast proteome. Taken together, the various LFQ methods and TMT quantification method optimized in this study showed high performance and various applications in quantitative proteomics.