Enhancement of plasmid-mediated stable gene expression by woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) in human embryonic kidney (HEK293) cells

Influence of random integration site on the expression of transgene in mammalian cells makes it a major challenge to achieve high productivity of recombinant proteins. Optimization of expression vector is one of the most popular strategies to resolve this problem. Among this, woodchuck hepatitis virus  post-transcriptional regulatory element (WPRE) is a possible enhancer of gene expression in mammalian  cells that promotes efficient export of unspliced (RNA) into the cytoplasm, as has been proved in  transient transfection. In this study, WPRE was evaluated for enhancing stable gene expression levels in two industrial cell lines, human embryonic kidney (HEK293) and CHO-S, using the enhanced green fluorescent protein (EGFP), prourokinase (pro-UK) and protein C (PC) as the reporter gene. Based on the mean fluorescence intensity (MFI), WPRE exerted a clear positive effect on gene expression in HEK293 cells with an increase of EGFP expression level by approximately 2.5- to 3-fold independent of the  promoter used in plasmid vector. In contrast, in Chinese hamster ovary (CHO)-S cells, only a marginal effect on plasmid-mediated EGFP expression by WPRE was observed. The measurable increase of EGFP expression at the protein level was paralleled by an increase of EGFP RNA. Further test of the effect of WPRE on plasmid-mediated gene expression with two therapeutic proteins showed substantial increase of stable pro-UK and PC expression only in HEK293 by about 2.2-fold and 6.1-fold, respectively. The data of PC expression levels obtained from the random HEK293 cell clones transfected with WPRE-containing or lacking vector further demonstrated the enhancement of stable plasmid-mediated gene expression by WPRE in HEK293 cells. These results in stable transfectants show the positive effect of WPRE on transgene expression is cell-type dependent and promoter-independent, and provide valuable information to improve vectors for efficient and stable gene expression in HEK293 cells.Key words: Mammalian cells, plasmid vector, stable gene expression, protein therapeutics, woodchuck hepatitis virus post-transcriptional regulatory element (WPRE)

A sample-position-autocorrection system with precision better than 1 \um~in angle-resolved photoemission experiments

We present the development of a high-precision sample-position-autocorrection system for photoemission experiments. A binocular vision method based on image pattern matching calculations was realized to track the sample position with an accuracy better than 1 \um, which was much smaller than the spot size of the incident laser. We illustrate the performance of the sample-position-autocorrection system with representative photoemission data on the topological insulator Bi$_2$Se$_3$ and an optimally-doped cuprate superconductor \Bi. Our method provides new possibilities for studying the temperature-dependent electronic structures in quantum materials by laser-based or spatially resolved photoemission systems with high precision and efficiency.Comment: 6 pages, 4 figure

Fractional Hardy-Sobolev type inequalities for half spaces and John domains

As our main result we prove a variant of the fractional Hardy-Sobolev-Maz'ya inequality for half spaces. This result contains a complete answer to a recent open question by Musina and Nazarov. In the proof we apply a new version of the fractional Hardy-Sobolev inequality that we establish also for more general unbounded John domains than half spaces

ChIP-less analysis of chromatin states

BACKGROUND: Histone post-translational modifications (PTMs) are key epigenetic regulators in chromatin-based processes. Increasing evidence suggests that vast combinations of PTMs exist within chromatin histones. These complex patterns, rather than individual PTMs, are thought to define functional chromatin states. However, the ability to interrogate combinatorial histone PTM patterns at the nucleosome level has been limited by the lack of direct molecular tools. RESULTS: Here we demonstrate an efficient, quantitative, antibody-free, chromatin immunoprecipitation-less (ChIP-less) method for interrogating diverse epigenetic states. At the heart of the workflow are recombinant chromatin reader domains, which target distinct chromatin states with combinatorial PTM patterns. Utilizing a newly designed combinatorial histone peptide microarray, we showed that three reader domains (ATRX-ADD, ING2-PHD and AIRE-PHD) displayed greater specificity towards combinatorial PTM patterns than corresponding commercial histone antibodies. Such specific recognitions were employed to develop a chromatin reader-based affinity enrichment platform (matrix-assisted reader chromatin capture, or MARCC). We successfully applied the reader-based platform to capture unique chromatin states, which were quantitatively profiled by mass spectrometry to reveal interconnections between nucleosomal histone PTMs. Specifically, a highly enriched signature that harbored H3K4me0, H3K9me2/3, H3K79me0 and H4K20me2/3 within the same nucleosome was identified from chromatin enriched by ATRX-ADD. This newly reported PTM combination was enriched in heterochromatin, as revealed by the associated DNA. CONCLUSIONS: Our results suggest the broad utility of recombinant reader domains as an enrichment tool specific to combinatorial PTM patterns, which are difficult to probe directly by antibody-based approaches. The reader affinity platform is compatible with several downstream analyses to investigate the physical coexistence of nucleosomal PTM states associated with specific genomic loci. Collectively, the reader-based workflow will greatly facilitate our understanding of how distinct chromatin states and reader domains function in gene regulatory mechanisms

Debridement of contaminated implants using air-polishing coupled with pH-responsive maximin H5-embedded metal-organic frameworks

The primary goal of peri-implantitis treatments remains the decontamination of implant surfaces exposed to polymicrobial biofilms and renders biocompatibility. In this study, we reported a synergistic strategy for the debridement and re-osteogenesis of contaminated titanium by using erythritol air abrasion (AA) coupled with an as-synthesized pH-responsive antimicrobial agent. Here, the anionic antibacterial peptide Maximin H5 C-terminally deaminated isoform (MH5C) was introduced into the Zeolitic Imidazolate Frameworks (ZIF-8) via a one-pot synthesis process. The formed MH5C@ZIF-8 nanoparticles (NPs) not only possessed suitable stability, but also guarantee the slow-release effect of MH5C. Antibacterial experiments revealed that MH5C@ZIF-8 NPs exhibited excellent antimicrobial abilities toward pathogenic bacteria of peri-implantitis, confirming ZIF-8 NPs as efficient nanoplatforms for delivering antibacterial peptide. To evaluate the comprehensive debridement efficiency, single-species as well as mixed-species biofilms were successively established on commercially used titanium surfaces and decontaminated with different methods: removed only by erythritol air abrasion, treated merely with MH5C@ZIF-8 NPs, or received both managements. The results demonstrated that only erythritol air abrasion accompanied with MH5C@ZIF-8 NPs at high concentrations eliminated almost all retained bacteria and impeded biofilm rehabilitation, while neither erythritol air abrasion nor MH5C@ZIF-8 NPs alone could achieve this. Subsequently, we evaluated the re-osteogenesis on previously contaminated surfaces which were treated with different debridement methods afterwards. We found that cell growth and osteogenic differentiation of bone marrow–derived mesenchymal stem cells (BMSCs) in the group received both treatments (AA + MH5C@ZIF-8) were higher than those in other groups. Our work emphasized the great potential of the synergistic therapy as a credible alternative for removing microorganisms and rendering re-osseointegration on contaminated implant surfaces, boding well for the comprehensive applications in peri-implantitis treatments

Ultrafast Switching from the Charge Density Wave Phase to a Metastable Metallic State in 1T-TiSe2_2

The ultrafast electronic structures of the charge density wave material 1T-TiSe$_2$ were investigated by high-resolution time- and angle-resolved photoemission spectroscopy. We found that the quasiparticle populations drove ultrafast electronic phase transitions in 1T-TiSe$_2$ within 100 fs after photoexcitation, and a metastable metallic state, which was significantly different from the equilibrium normal phase, was evidenced far below the charge density wave transition temperature. Detailed time- and pump-fluence-dependent experiments revealed that the photoinduced metastable metallic state was a result of the halted motion of the atoms through the coherent electron-phonon coupling process, and the lifetime of this state was prolonged to picoseconds with the highest pump fluence used in this study. Ultrafast electronic dynamics were well captured by the time-dependent Ginzburg-Landau model. Our work demonstrates a mechanism for realizing novel electronic states by photoinducing coherent motion of atoms in the lattice.Comment: 13 Pages, 10 figure

Memory Acquisition and Retrieval Impact Different Epigenetic Processes that Regulate Gene Expression

Background: A fundamental question in neuroscience is how memories are stored and retrieved in the brain. Long-term memory formation requires transcription, translation and epigenetic processes that control gene expression. Thus, characterizing genome-wide the transcriptional changes that occur after memory acquisition and retrieval is of broad interest and importance. Genome-wide technologies are commonly used to interrogate transcriptional changes in discovery-based approaches. Their ability to increase scientific insight beyond traditional candidate gene approaches, however, is usually hindered by batch effects and other sources of unwanted variation, which are particularly hard to control in the study of brain and behavior. Results: We examined genome-wide gene expression after contextual conditioning in the mouse hippocampus, a brain region essential for learning and memory, at all the time-points in which inhibiting transcription has been shown to impair memory formation. We show that most of the variance in gene expression is not due to conditioning and that by removing unwanted variance through additional normalization we are able provide novel biological insights. In particular, we show that genes downregulated by memory acquisition and retrieval impact different functions: chromatin assembly and RNA processing, respectively. Levels of histone 2A variant H2AB are reduced only following acquisition, a finding we confirmed using quantitative proteomics. On the other hand, splicing factor Rbfox1 and NMDA receptor-dependent microRNA miR-219 are only downregulated after retrieval, accompanied by an increase in protein levels of miR-219 target CAMKIIγ. Conclusions: We provide a thorough characterization of coding and non-coding gene expression during long-term memory formation. We demonstrate that unwanted variance dominates the signal in transcriptional studies of learning and memory and introduce the removal of unwanted variance through normalization as a necessary step for the analysis of genome-wide transcriptional studies in the context of brain and behavior. We show for the first time that histone variants are downregulated after memory acquisition, and splicing factors and microRNAs after memory retrieval. Our results provide mechanistic insights into the molecular basis of cognition by highlighting the differential involvement of epigenetic mechanisms, such as histone variants and post-transcriptional RNA regulation, after acquisition and retrieval of memory

Optical High Content Nanoscopy of Epigenetic Marks Decodes Phenotypic Divergence in Stem Cells

While distinct stem cell phenotypes follow global changes in chromatin marks, single-cell chromatin technologies are unable to resolve or predict stem cell fates. We propose the first such use of optical high content nanoscopy of histone epigenetic marks (epi-marks) in stem cells to classify emergent cell states. By combining nanoscopy with epi-mark textural image informatics, we developed a novel approach, termed EDICTS (Epi-mark Descriptor Imaging of Cell Transitional States), to discern chromatin organizational changes, demarcate lineage gradations across a range of stem cell types and robustly track lineage restriction kinetics. We demonstrate the utility of EDICTS by predicting the lineage progression of stem cells cultured on biomaterial substrates with graded nanotopographies and mechanical stiffness, thus parsing the role of specific biophysical cues as sensitive epigenetic drivers. We also demonstrate the unique power of EDICTS to resolve cellular states based on epi-marks that cannot be detected via mass spectrometry based methods for quantifying the abundance of histone posttranslational modifications. Overall, EDICTS represents a powerful new methodology to predict single cell lineage decisions by integrating high content super-resolution nanoscopy and imaging informatics of the nuclear organization of epi-marks.National Institutes of Health (U.S.) (Grant GM110174

Smyd3 regulates cancer cell phenotypes and catalyzes histone H4 lysine 5 methylation

Smyd3 is a lysine methyltransferase implicated in chromatin and cancer regulation. Here we show that Smyd3 catalyzes histone H4 methylation at lysine 5 (H4K5me). This novel histone methylation mark is detected in diverse cell types and its formation is attenuated by depletion of Smyd3 protein. Further, Smyd3-driven cancer cell phenotypes require its enzymatic activity. Thus, Smyd3, via H4K5 methylation, provides a potential new link between chromatin dynamics and neoplastic disease