Genetic Tools for Generating Mutant Clones and Somatic Mutagenesis in Mice.

<p>Genetic Tools for Generating Mutant Clones and Somatic Mutagenesis in Mice.</p

Somatic phenotypes like cancer can be modeled and genetically dissected with transposon mutagenesis.

<p>Potential oncogenic pathway to be interrogated with candidate oncogene X and effector Y in red (left). Depiction of <i>PB</i> transposon construct for verifying oncogene X (center). Yellow arrows detail transposon arms. Promoters are depicted by blue pointed boxes. Gene X is indicated by red box and luciferase marker is indicated by green box. To test if effector Y is involved in the oncogenic pathway, an shRNA cassette to knockdown gene Y is represented by the red box (right). The transposons are co-transfected or electroporated with <i>PBase</i> (lower yellow box) to stably integrate the transposon construct into the mouse cells. The green cells in the mouse indicate luciferase positive cells expressing the transposed construct, which are monitored for the tumor formation.</p

Automated Capillary Isoelectric Focusing-Mass Spectrometry with Ultrahigh Resolution for Characterizing Microheterogeneity and Isoelectric Points of Intact Protein Complexes

Protein complexes are the functional machines in the cell and are heterogeneous due to protein sequence variations and post-translational modifications (PTMs). Here, we present an automated nondenaturing capillary isoelectric focusing-mass spectrometry (ncIEF-MS) methodology for uncovering the microheterogeneity of intact protein complexes. The method exhibited superior separation resolution for protein complexes than conventional native capillary zone electrophoresis (nCZE-MS). In our study, ncIEF-MS achieved liquid-phase separations and MS characterization of seven different forms of a streptavidin homotetramer with variations of N-terminal methionine removal, acetylation, and formylation and four forms of the carbonic anhydrase–zinc complex arising from variations of PTMs (succinimide, deamidation, etc.). In addition, ncIEF-MS resolved different states of an interchain cysteine-linked antibody–drug conjugate (ADC1) as a new class of anticancer therapeutic agents that bears a distribution of varied drug-to-antibody ratio (DAR) species. More importantly, ncIEF-MS enabled precise measurements of isoelectric points (pIs) of protein complexes, which reflect the surface electrostatic properties of protein complexes. We studied how protein sequence variations/PTMs modulate the pIs of protein complexes and how drug loading affects the pIs of antibodies. We discovered that keeping the N-terminal methionine residue of one subunit of the streptavidin homotetramer decreased its pI by 0.1, adding one acetyl group onto the streptavidin homotetramer reduced its pI by nearly 0.4, incorporating one formyl group onto the streptavidin homotetramer reduced its pI by around 0.3, and loading two more drug molecules on one ADC1 molecule increased its pI by 0.1. The data render the ncIEF-MS method a valuable tool for delineating protein complexes

Screening for phenotypes in humanized mice with patient-derived IPS cells.

<p>IPS cells are first created from a patient. A mutator transposon containing mutagenic elements (red box) and a GFP marker (green box) and an inducible <i>PBase</i> construct (utilizing the Cre-ER/lox or Tet system) is introduced into patient-derived IPS cells. Green cells indicate GFP expression from the stably integrated mutator transposon(s). The cells are then introduced into the mouse tissue by injection (syringe). Next, transposase activity is induced, which mobilizes the mutagenic transposon, resulting in insertional mutation. Finally, the mice are screened for the desired disease or developmental phenotype.</p

Three-Enzyme Cascade Bioreactor for Rapid Digestion of Genomic DNA into Single Nucleosides

Structure-based DNA modification analysis provides accurate and important information on genomic DNA changes from epigenetic modifications to various DNA lesions. However, genomic DNA strands are often required to be efficiently digested into single nucleosides. It is an arduous task because of the involvement of multiple enzymes with different catalytic acitivities. Here we constructed a three-enzyme cascade capillary monolithic bioreactor that consists of immobilized deoxyribonuclease I (DNase I), snake venom phosphodiesterase (SVP), and alkaline phosphatase (ALPase). By the use of this cascade capillary bioreactor, genomic DNA can be efficiently digested into single nucleosides with an increasing rate of ∼20 folds. The improvement is mainly attributed to dramatically increase enzymatic capacity and activity. With a designed macro-porous structure, genomic DNA of 5–30 Kb (∼1.6–10 million Daltons) can be directly passed through the bioreactor simply by hand pushing or a low-pressure microinjection pump. By coupling with liquid chromatography-tandem mass spectrometry (LC-MS/MS), we further developed a sensitive assay for detection of an oxidative stress biomarker 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in DNA. The proposed three-enzyme cascade bioreactor is also potentially applicable for fast identification and quantitative detection of other lesions and modifications in genomic DNA

Nickel-Catalyzed Suzuki–Miyaura Reaction of Aryl Fluorides

Two protocols for the nickel-catalyzed cross-coupling of aryl fluorides with aryl boronic esters have been developed. The first employs metal fluoride cocatalysts, such as ZrF4 and TiF4, which enable Suzuki–Miyaura reactions of aryl fluorides bearing electron-withdrawing (ketones, esters, and CF3), aryl and alkenyl groups as well as those comprising fused aromatic rings, such as fluoronaphthalenes and fluoroquinolines. The second protocol employs aryl fluorides bearing ortho-directing groups, which facilitate the difficult C–F bond activation process via cyclometalation. N-heterocycles, such as pyridines, quinolines, pyrazoles, and oxazolines, can successfully promote cross-coupling with an array of organoboronic esters. A study into the substituent effects with respect to both coupling components has provided fundamental insights into the mechanism of the nickel-catalyzed cross-coupling of aryl fluorides

Coupling High-Field Asymmetric Waveform Ion Mobility Spectrometry with Capillary Zone Electrophoresis-Tandem Mass Spectrometry for Top-Down Proteomics

Capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) has emerged as an essential technique for top-down proteomics (TDP), providing superior separation efficiency and high detection sensitivity for proteoform analysis. Here, we aimed to further enhance the performance of CZE-MS/MS for TDP via coupling online gas-phase proteoform fractionation using high-field asymmetric waveform ion mobility spectrometry (FAIMS). When the compensation voltage (CV) of FAIMS was changed from −50 to 30 V, the median mass of identified proteoforms increased from less than 10 kDa to about 30 kDa, suggesting that FAIMS can efficiently fractionate proteoforms by their size. CZE-FAIMS-MS/MS boosted the number of proteoform identifications from a yeast sample by nearly 3-fold relative to CZE-MS/MS alone. It particularly benefited the identification of relatively large proteoforms, improving the number of proteoforms in a mass range of 20–45 kDa by 6-fold compared to CZE-MS/MS alone. FAIMS fractionation gained nearly 20-fold better signal-to-noise ratios of randomly selected proteoforms than no FAIMS. We expect that CZE-FAIMS-MS/MS will be a useful tool for further advancing the sensitivity and coverage of TDP. This work shows the first example of coupling CE with ion mobility spectrometry (IMS) for TDP

Coupling High-Field Asymmetric Waveform Ion Mobility Spectrometry with Capillary Zone Electrophoresis-Tandem Mass Spectrometry for Top-Down Proteomics

Capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) has emerged as an essential technique for top-down proteomics (TDP), providing superior separation efficiency and high detection sensitivity for proteoform analysis. Here, we aimed to further enhance the performance of CZE-MS/MS for TDP via coupling online gas-phase proteoform fractionation using high-field asymmetric waveform ion mobility spectrometry (FAIMS). When the compensation voltage (CV) of FAIMS was changed from −50 to 30 V, the median mass of identified proteoforms increased from less than 10 kDa to about 30 kDa, suggesting that FAIMS can efficiently fractionate proteoforms by their size. CZE-FAIMS-MS/MS boosted the number of proteoform identifications from a yeast sample by nearly 3-fold relative to CZE-MS/MS alone. It particularly benefited the identification of relatively large proteoforms, improving the number of proteoforms in a mass range of 20–45 kDa by 6-fold compared to CZE-MS/MS alone. FAIMS fractionation gained nearly 20-fold better signal-to-noise ratios of randomly selected proteoforms than no FAIMS. We expect that CZE-FAIMS-MS/MS will be a useful tool for further advancing the sensitivity and coverage of TDP. This work shows the first example of coupling CE with ion mobility spectrometry (IMS) for TDP

POSH regulates Hippo signaling through ubiquitin-mediated expanded degradation

The Hippo signaling pathway is a master regulator of organ growth, tissue homeostasis, and tumorigenesis. The activity of the Hippo pathway is controlled by various upstream components, including Expanded (Ex), but the precise molecular mechanism of how Ex is regulated remains poorly understood. Here we identify Plenty of SH3s (POSH), an E3 ubiquitin ligase, as a key component of Hippo signaling in Drosophila. POSH overexpression synergizes with loss of Kibra to induce overgrowth and up-regulation of Hippo pathway target genes. Furthermore, knockdown of POSH impedes dextran sulfate sodium-induced Yorkie-dependent intestinal stem cell renewal, suggesting a physiological role of POSH in modulating Hippo signaling. Mechanistically, POSH binds to the C-terminal of Ex and is essential for the Crumbs-induced ubiquitination and degradation of Ex. Our findings establish POSH as a crucial regulator that integrates the signal from the cell surface to negatively regulate Ex-mediated Hippo activation in Drosophila

MXene-Based Mixed Conductor Interphase for Dendrite-Free Flexible Al Organic Battery

Al batteries are promising post-Li battery technologies for large-scale energy storage applications owing to their low cost and high theoretical capacity. However, one of the challenges that hinder their development is the unsatisfactory plating/stripping of the Al metal anode. To circumvent this issue, an ultrathin MXene layer is constructed on the surface of Al by in situ chemical reactions at room temperature. The as-prepared flexible MXene film acts like armor to protect the Al-metal by its high ionic conductivity and high mechanical flexibility. The MXene endow the Al anode with a long cyclic life of more than 5000 h at ultrahigh current density of 50 mA cm–2 for Al//Al batteries and a retention of 100% over 200 cycles for 355 Wh kg–1 PTO//Al batteries. This work provides fresh insights into the formation and regulation of stable electrode–electrolyte interfaces as well as effective strategies for improving Al metal batteries